In this issue

M
ore than 1,400 bacterial, viral and parasitic species are
known to cause human disease. Understanding how these
pathogens colonize, survive and inflict disease remains
one of the key goals of microbiology. This, the first issue
of 2008, features several Reviews that discuss microbial virulence.
On page 79, John Boothroyd and Jean-Francois Dubremetz discuss
the specialized rhoptry organelle, which exists in apicomplexan parasites
such as Toxoplasma gondii. In T. gondii, recent findings indicate that these
organelles contain the molecular machinery that enables the parasite to
affect host gene expression and co-opt host functions.
▶ cover: ‘A to B’ by George Marshall, inspired by Dealing with bacteria, Samuel Miller and colleagues discuss the
the Review on p28.
mechanisms that enable Salmonellae to interact with, and manipulate, host
cells (page 53). Specifically, they focus on the interplay between various
bacterial and host proteins that enables the invasion of epithelial cells,
stimulation and repression of signalling cascades, sensing of the intracellular
environment and establishment of a niche for intracellular replication.
So, how can we harness our understanding of microbial virulence to stem
david o’connell susan jones the continuing rise in infectious-disease-related morbidity and mortality?
Antibiotic-based strategies, which typically target bacterial viability, have
resulted in widespread resistance — meticillin-resistant Staphylococcus
aureus infections reached epidemic levels last autumn in some parts of the
United States. Perhaps rather than targeting bacteria for eradication, we
sheilagh molloy sharon Ahmad
should focus instead on de-clawing pathogens by inhibiting the virulence
mechanisms that promote infection or that cause disease symptoms. On
page 17, Scott Hultgren and colleagues highlight various bacterial virulence
mechanisms that could be targeted and consider the recent efforts towards,
and the remaining challenges that face, antivirulence-based drug discovery.

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nature reviews | microbiology volume 6 | january 2008 | 

© 2008 Nature Publishing Group
Editorial
The value of vaccines
Our ability to control infectious diseases is continuously being eroded by antimicrobial
resistance, the decline in industrial antibiotic development and increasing development
timelines from discovery to market. There has never been a better time to rediscover the
value of vaccination.
As a preventative strategy in the fight against infectious
diseases, vaccination is considered to be the most cost-
effective medical intervention. Indeed, in many devel-
oped countries, measles, mumps, rubella, hepatitis B,
Haemophilus influenzae type b, tetanus, pertussis and
diphtheria have been controlled or nearly eliminated
as chief causes of morbidity and mortality by the use of
vaccines. Yet, despite their role in delivering many of the
successes that have been achieved in infectious disease
control, there seems to be relatively little enthusiasm
for vaccine development among those who have the
capability for vaccine development and production.
There are a number of contributing factors to this lack
of enthusiasm, most of which are linked to economics.
The simple truth is that the direct economic value that is
associated with vaccines is negligible compared with that
of pharmaceutical drugs. Globally, potential vaccine sales
are approximately US$6 billion per year. Although the
vaccines sector is out-performing the rest of the industry
in terms of revenue growth, it still represents less than
2% of the worldwide pharmaceutical market. Because of
the low return for their investment, high regulatory costs,
uncertain market conditions and exposure to legal liabil-
ity, most pharmaceutical manufacturers do not consider
the development and production of vaccines as an attrac-
tive business opportunity. For example, over the past
30 years, the number of companies that distribute vaccines
in the United States has decreased from 30 to 5, a situa-
tion that has directly contributed to serious shortages of
influenza, tetanus–diphtheria, measles–mumps–rubella,
pneumococcal, meningococcal and other vaccines. Not
surprisingly, there is even less incentive for the vaccine
the direct industry to develop new vaccines against diseases that
economic value are largely limited to the developing world.
that is associated Governments do have a number of well-documented
and under-used tools at their disposal to make vaccines
with vaccines more attractive to industry. These options include: tax
is negligible breaks or subsidies to reduce research and development
compared expenses; extending patent protection for intellectual
with that of property that is related to vaccines of public health impor-
tance; working with industry and others to find ways to
pharmaceutical reduce the costs of meeting regulatory requirements; and
drugs. allowing tiered pricing whereby vaccines can be sold at
 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
higher prices in developed countries and lower prices in
under-developed countries. It should also be possible to
decrease liability risks (by measures such as the Vaccine
Injury Compensation Program in the United States) and
protect manufacturers from lawsuits that are related to
the unanticipated adverse effects of a properly manufac-
tured, safe and effective vaccine. Ultimately, however, an
economic solution is required. There have been numer-
ous demonstrations of the cost-effectiveness of immuni-
zation. Attributing the true economic value to vaccines
— both real and intangible — will create a self-sustaining
system to ensure the development and adequate supply
of vaccines to those that need them most.
But how can governments be mobilized into action?
If the ever-increasing threat of a public health calamity
does not provide the necessary incentive, the promise of
success might do the trick. An example that illustrates the
effectiveness of a global approach to vaccination is the
campaign for the eradication of poliomyelitis. Launched
in 1985 for South America, it was taken up by the World
Assembly, which in 1988 committed to the global eradi-
cation of poliovirus by the year 2000. Progress towards
eradication of the virus has been fast. In 1988, 125 coun-
tries in 5 continents reported endemic poliovirus but by
2003 only 6 polio-endemic countries were reported and,
officially, only 4 remain: India, Pakistan, Afghanistan and
Nigeria. So, although the 2000 deadline passed with the
incidence of disease stalled at around 2,000 cases per year,
and there have been setbacks, including a recent outbreak
in Nigeria, the initiative is an unequivocal example of
how a globally coordinated effort can, within a short
period of time, reduce the incidence of an infectious dis-
ease by more than 99.9%. The elimination of the disease
from the remaining endemic regions is now achievable,
leaving the welcome problem of whether, and how long,
vaccination should be continued against a disease that
no longer exists.
In their efforts to control infectious diseases, govern-
ments and non-governmental organizations need look
no further than the poliomyelitis eradication campaign,
both for their inspiration and justification in taking that
first important step — placing vaccination back where it
belongs, at the top of the global public health agenda.

hi v
Infection, superinfection
and (lack of) protection
Superinfection with HIV-1 occurs
when an individual who is already
infected with one strain of HIV-1
acquires a second strain from
a different partner. Because a
re-infection event suggests that
the immune response generated
against the original infection is
not sufficient to protect against
later exposures, there are obvious
implications for HIV-1 vaccine
design. However, despite the
potential importance of superinfec-
tion in HIV disease and vaccine
development, there is uncertainty
regarding its incidence and timing.
Now, an in-depth, population-level
assessment of HIV-1 superinfection,
published in PLoS Pathogens, con-
firms that natural HIV-1 infection
does not always elicit a protective
immune response, and that this lack
of protection is largely independent
0 1
Time (years post-infection)
Superinfection with an HIV-1 virus from a second partner (pink) can occur several years after an initial HIV-1 infection (green). At this time
(see arrow), cytotoxic T-cell responses (CTL) and neutralizing antibody responses (NTAb) to the first infection have had time to develop.
Figure redrawn, with permission, from one kindly provided by Julie Overbaugh, Fred Hutchinson CancerNatureResearchReviews
nature reviews | microbiology volume 6 | january 2008
of the timing of re-exposure and the
relatedness of the virus strains.
Prior to this study, approximately
twenty cases of HIV-1 superinfection
had been reported in the literature,
which suggested that natural infec-
tion does not always generate a pro-
tective immune response. However,
if researchers are to accurately assess
the impact of superinfection on the
success of an HIV-1 vaccine, ques-
tions need to be answered about
the frequency of superinfection and
whether this process is restricted
to the times in infection when an
HIV‑1-specific immune response has
not yet developed.
To address these issues, Piantadosi
and colleagues screened a cohort of
high-risk Kenyan women for HIV-1
superinfection by comparing 2 partial
gene sequences (gag and envelope) over
a 5-year period, beginning at the time
2 3
© 2008 Nature Publishing Group
Research highlights
of primary infection. Analysing more
than one region of the HIV-1 genomes
allowed the authors to detect cases in
which the initial and superinfecting
viruses had recombined, a frequent
event during HIV-1 replication. By
analysing the proviral sequences from
36 women infected with HIV-1, seven
cases of superinfection were detected,
including 3 cases in which both
…this lack viruses belonged to the same HIV-1
of protection subtype; this finding indicates that
is largely closely related viruses do not confer
protection. The authors also showed
independent of
that in 5 of the 7 cases, infection with
the timing of re- the second virus occurred over 1 year
exposure and the after the initial infection, and, in some
relatedness of the cases, 5 years after initial infection. In
virus strains. other words, superinfection occurred
after the host immune response to
the first HIV-1 strain should have had
time to develop and mature.
This study clearly demonstrates
that HIV-positive individuals are at
continued risk of acquiring a second
HIV infection. Indeed, if the lack
of protection against re-infection
is shared by at least a proportion
of all HIV-infected individuals, the
potential value of an HIV-1 vaccine
that attempts to mimic the immune
response to natural infection is called
into question. Follow-up studies to
compare the immune responses of
those individuals that become super-
infected with those that do not, and
CTL deciphering the contribution of these
NtAb responses to immune protection, will
HIV-1 RNA be the focus of future research.
David O’Connell
4 5 ORIGINAL RESEARCH PAPER Piantadosi, A.
et al. Chronic HIV-1 infection frequently fails to
protect against superinfection. PLoS Pathog. 3,
e177 (2007)
| Microbiology
Center, Washington, USA.
 Research highlights

MicroBIAL Physiology

How low can you grow?

Removal of the greenhouse gas
methane by methanotrophic bacteria
is an important climatic process. All
methanotrophs characterized so far
are meso- or thermophilic members
of the phylum Proteobacteria. Now,
two papers published in Nature
report the cultivation, characteriza-
tion and draft genome analysis of
two new methanotrophs from the
phylum Verrucomicrobia that are
the most acidophilic methanotrophs
ever studied.
Some environments that are
rich in methane are also extremely
acidic, so two groups of researchers
set out to investigate whether novel
methanotrophs thrive in these
conditions. PCR primer sets for
the conserved pmoA gene, which
encodes a subunit of the particulate
form of methane monooxygenase,
have been used routinely to sur-
vey numerous environments for
methanotrophs. Pol et al. extracted
DNA from a hot, acidic, methane-
producing fumarole in the Solfatara,
Italy, and used primers for pmoA to
construct an environmental clone
library. They found two classes
of pmoA sequences in the library
— gammaproteobacterial-like
sequences and a divergent set of
pmoA homologues. Intrigued by
these unusual pmoA sequences,
they isolated a strain into pure
culture that had a divergent pmoA
sequence that was similar to those
present in the clone library and
a 16S rRNA sequence that was
typical of Verrucomicrobia, and
named it SolV (provisional name
Acidimethylosilex fumarolicum).
In a parallel study, Dunfield
et al. analysed 16S rRNA genes of
bacteria in a sample from Hell’s Gate,
an acidic geothermal site in New
Zealand that is rich in methane, and
identified novel Verrucomicrobia
sequence signatures that were
unrelated to any cultured bacteria.
A strain named Verrucomicrobia
isolate V4 (provisional name
Methylokorus infernorum) was
isolated into pure culture from this
sample by supplying methane as the
sole carbon and energy source. Both
SolV and V4 have one striking phe-
notype: V4 can grow at pH 1.0 and
SolV can grow at pH 0.8. This is the
first time that extremely acidophilic
methanotrophs have been described.
Draft genomes of both new species
were assembled and scrutinized for
insights into their physiologies; both
are remarkable in that they have three
pmoCAB operons that are completely
different. Most proteobacterial
methanotrophs typically encode up
to three similar pmoCAB operons.
Phylogenetic analyses of the three
pmoA alleles encoded by V4 and
those encoded by SolV indicate that
these acidophilic lineages diverged
from Proteobacteria a long time
ago, and exclude the possiblity
that Verrucomicrobia species have
acquired the pmoA gene by a recent
horizontal-gene-transfer event.
Inspection of the genomes indicated
that both species might also use novel
methylotrophic pathways.
These studies have provided
an important step forward in the
understanding of the physiology and
diversity of methane consumption by
bacteria in natural environments.
Susan Jones
ORIGINAL RESEARCH PAPERS Pol, A. et al.
Methanotrophy below pH1 by a new
Verrucomicrobia species. Nature 14 Nov 2007
(doi 10.1038/nature06222) | Dunfield, P. F. et al.
Methane oxidation by an extremely acidophilic
bacterium of the phylum Verrucomicrobia.
Nature 14 Nov 2007 (doi 10.1038/nature06411)

nature reviews | microbiology volume 6 | january 2008

© 2008 Nature Publishing Group

RNA
Extending the network of sRNA control
Mechanisms by which the
small non-coding RNAs
(sRNAs) of Gram-negative
bacteria regulate multiple
mRNA targets have proved
difficult to pin down, owing
to the limited complementarity
between sRNAs and target mRNAs.
In a paper published in Genes &
Development, Jörg Vogel and
colleagues report that GcvB, a
200-nucleotide sRNA, directly
regulates 7 mRNAs that encode
periplasmic peptide and amino-
acid transport proteins in
Salmonella enterica serovar
Typhimurium (S. typhimurium).
The GcvB sRNA represses
translation of the dppA and oppA pep-
tide transporter genes in nutrient-rich
conditions. Intriguingly, gcvB mutants
are pleiotropic, and computational
analyses indicated that GcvB might
have numerous target genes, which
led Vogel and colleagues to investigate
how the GcvB sRNA functions.
RNA-structure probing pinpointed a
29-nucleotide G- and U-rich linker
region in GcvB, named R1, that
interacts with sequences that span the
ribosome binding site (RBS) of both
peptide transporter genes. Strikingly,
deletion of R1 destroyed the ability of
GcvB to repress dppA and oppA.
Bioinformatic searches revealed
that R1 is well-conserved among
the gcvB genes of Pasteurella spp.,
Vibrio spp. and many enterobacte-
ria. By combining scrutiny of the
nature reviews | microbiology volume 6 | january 2008
© 2008 Nature Publishing Group
Research highlights
periplasmic-protein profiles of an
S. typhimurium strain that is over-
expressing gcvB with an in silico
screen for binding partners of R1,
Vogel and co-workers identifed five
additional candidate GcvB targets
that encode periplasmic amino-acid
transport proteins — gltI, livJ, livK,
argT and STM4351. The predicted
GcvB-binding sites in these genes
were not conserved, but were rich in
C and A residues.
Biochemical assays revealed that
GcvB functions as a sequence-specific
inhibitor of translation, but the details
of inhibition vary among the different
target mRNAs. GcvB binds to the RBS
of dppA and oppA mRNAs to repress
translation by occlusion of the
ribosome. Surprisingly, GcvB
binds to sequences upstream
of the RBS of other regulated
genes, but still prevents the 30S
ribosome from docking onto the
mRNA to initiate translation.
Fusion of the 5′-untranslated
region of gltI, which contains
the GcvB-docking site,
with the unrelated E. coli
ompR gene conferred GcvB-
mediated repression to ompR,
proving that GcvB can regulate any
mRNA that contains a cognate C-
and A‑rich R1-binding motif. The
precise mechanism of this
novel type of translational
control remains unclear.
Other studies have shown
that an sRNA (514 nucleotides)
named RNAIII functions to
repress multiple virulence factors
in the Gram-positive pathogen
Staphylococcus aureus by comple-
mentary base-pairing with the RBS
of target-gene mRNAs. Together,
these studies show that specific
interactions between sRNAs and
mRNAs in both Gram-positive
and Gram-negative bacteria can
regulate multiple genes.
Susan Jones
ORIGINAL RESEARCH PAPER Sharma, C. M.,
Darfeuille, F., Plantinga, T. H. & Vogel, J.
A small RNA regulates multiple ABC transporter
mRNAs by targeting C/A-rich elements inside
and upstream of ribosome-binding sites.
Genes Dev. 21, 2804–2817 (2007)

Host response
IgA — peacemaker in the gut
Using a novel gnotobiotic mouse
model in which the diverse gut
microbiota is reduced to a single
bacterial species, and the antibody
repertoire to a single monoclonal
immunoglobulin A (IgA) antibody
that is directed against the bacteri-
um’s capsular polysaccharide, Jeffrey
Gordon and colleagues provide
evidence that supports a role for IgA
in the gut as a mediator of tolerance.
The IgA antibody response has a
key role in establishing and maintain-
ing a non-inflammatory relationship
between the host and microbiota
in the gut. Germ-free mice that are
colonized with normal gut microbiota
develop bacteria-specific IgA antibody
responses, but the effects of these
responses on the biology of the host
and microbiota are not well defined.
Peterson et al. developed a gnotobiotic
mouse model to study these effects.
As their model symbiont, they
chose the bacterium Bacteroides
nature reviews | microbiology volume 6 | january 2008
© 2008 Nature Publishing Group
Research highlights
thetaiotaomicron, which is a promi-
nent, obligately anaerobic, Gram-
negative member of the human
distal intestinal ecosystem that also
efficiently colonizes the intestines of
adult germ-free C57BL/6J mice.
B. thetaiotaomicron was introduced
into germ-free, recombination-
activating-gene-1-deficient (Rag1–/–)
mice (which lack mature B and T
cells) or germ-free Rag1–/– mice that
had been injected under their dorsal
skin with B. thetaiotaomicron-primed
IgA-producing hybridoma cells.
An inverse relationship was found
between IgA antibody levels and
the levels of its B. thetaiotaomicron
epitope in the intestinal lumen
— that is, infected mice with IgA
secreted by the hybridoma cells
had lower levels of epitope expres-
sion than mice without IgA. In the
absence of IgA, B. thetaiotaomicron
elicited a more robust innate
immune oxidative response, and
adapted to this response by induc-
ing genes that are involved in the
metabolism of oxidative products
of the host response. The presence
of IgA, however, reduced intestinal
pro-inflammatory signalling (by
downregulating signal transducer
and activator of transcription 3 and
interferon regulatory factor 8, for
example), and bacterial epitope
expression.
Therefore, these results suggest
that it is the IgA antibody response
that establishes a quiescent relation-
ship between B. thetaiotaomicron
and its host. They are also consistent
with a model in which a set of adap-
tations that involve both the symbi-
ont and its host lead to co-evolved
homeostasis.
Sharon Ahmad
ORIGINAL RESEARCH PAPER Peterson, D. A.
et al. IgA response to symbiotic bacteria as a
mediator of gut homeostasis. Cell Host Microbe
2, 328–339 (2007)
 Research highlights

in brief
b a c t e r i a l pat h o g e n e s i s
Invasive and adherent bacterial pathogens co-opt
host clathrin for infection
Veiga, E. et al. Cell Host & Microbe 2, 340–351 (2007)
Although many viruses enter host cells by clathrin-mediated
endocytosis, it was thought that clathrin-coated vesicles were
too small to internalize bacteria. So, it was a surprise when
Listeria monocytogenes was shown to enter host cells by a
clathrin-dependent mechanism. Building on this previously
published finding, Veiga et al. investigated whether clathrin
is required for the entry of other pathogens. They found
that bacteria that enter cells after interactions with specific
receptors, such as Staphylococcus aureus, require clathrin for
entry, whereas those that inject effectors into host cells to
facilitate their own entry by altering the host cytoskeleton,
such as Shigella flexneri, do not. Clathrin was also required
to assemble pedestals beneath adherent enteropathogenic
Escherichia coli, which remain extracellular.

molecular ecology
Mutational activation of niche-specific genes provides
insight into regulatory networks and bacterial
function in a complex environment
Giddens, S. R. et al. Proc. Natl Acad. Sci. USA 104, 18247–18252 (2007)
The identification of environment-induced loci (EIL) is hampered
by a lack of discernable phenotypes in the laboratory when EIL
are mutated. Giddens et al. describe a new, broadly applicable
method — SpyVet (suppressor analysis with in vivo expression
technology) — to identify regulatory loci that control EIL in the
rhizosphere bacterium Pseudomonas fluorescens. Promoters of
EIL are fused to dapB (required for lysine biosynthesis), rendering
strains prototrophic in the rhizosphere, where EIL are expressed,
but auxotrophic in minimal growth media, where EIL are not
expressed. Transposon mutagenesis, coupled with a simple
screen for prototrophy in the laboratory, can quickly identify
genes that regulate EIL–dapB fusions. A secondary screen for
other members of the regulatory hierarchy was also feasible,
which enabled these researchers to identify a regulatory
network of seven regulators.

b ac t e r i a l s e c r e t i o n
A minimal Tat system from a Gram-positive organism:
a bifunctional TatA subunit participates in discrete
TatAC and TatA complexes
Barnett, J. P. et al. J. Biol. Chem. 26 Nov 2007 (doi 10.1074/jbc.
M708134200)
The Tat (twin-arginine) secretion system transports fully folded
proteins across bacterial membranes. Gram-negative bacteria
encode three subunits (TatABC) that are essential for secretion.
TatABC form a substrate-binding complex that is thought to
recruit a separate and heterogeneous TatA complex, which can
form a translocation pore. Bacillus subtilis has three TatA and
two TatC variants. However, it lacks tatB, as do all Gram-positive
bacteria. The B. subtilis tatAd gene complemented a tatB mutant
of Escherichia coli, indicating that tatAd can replace TatA and
TatB. Importantly, a B. subtilis TatAdCd system secreted a Tat
substrate upon expression in E. coli, even though the TatAd
complex is discrete, in contrast to the heterogeneous E. coli TatA
complex. Direct experiments in B. subtilis are now needed to
resolve the current controversy over how Tat secretes proteins.

nature reviews | microbiology volume 6 | january 2008

© 2008 Nature Publishing Group

b ac t erial d e v elop m en t
Moving in the right direction
New data published in a recent issue
of Molecular Microbiology might
have finally solved the controversy
over the function of SpoIIIE dur-
ing sporulation in Bacillus subtilis.
During sporulation, B. subtilis
undergoes an asymmetric cell
division that generates two cells of
unequal size — a small forespore
and a larger mother cell — and a
chromosome must be accurately
segregated into each cell. The asym-
metric-division septum is formed
before chromosome segregation is
completed, pinching one of the repli-
cated chromosomes into a small lobe,
which constitutes ~30% of the genome
and is present in the forespore, and
a large lobe, which constitutes ~70%
of the genome and is present in the
mother cell. To ensure successful
completion of chromosome segrega-
tion, the trapped large lobe must be
translocated across the septum from
the mother cell into the forespore.
Chromosome segregation during
sporulation requires the double-
stranded DNA translocase SpoIIIE,
a protein that is related to the FtsK
translocase of non-spore-forming
bacteria. In recent years, results from
different laboratories have supported
two models of SpoIIIE/FtsK func-
tion. In the first, which is based on
single-molecule studies of FtsK, the
proteins function as reversible DNA
translocases, and the polarity of the
translocated DNA is determined by
the DNA substrate. In the second,
SpoIIIE functions as a DNA exporter,
and the polarity of the translocated
DNA is determined by the cell-
specific assembly of a stable SpoIIIE
nature reviews | microbiology volume 6 | january 2008
© 2008 Nature Publishing Group
Research highlights
translocation
complex. In this
work, Eric Becker and Kit Pogliano
set out to determine which of these
models is correct.
They began by devising a method
that would allow them to tag SpoIIIE
specifically in the mother cell and the
forespore. To do so, the authors made
use of the high-affinity interaction
between the leucine zipper domains
of cFos and cJun — the leucine zip-
per domain of cFos was fused to the
carboxyl terminus of SpoIIIE and the
leucine zipper domain of cJun was
fused to the amino terminus of green
fluorescent protein (GFP). Cell-
specific promoters were used to
express this GFP fusion protein
in either the mother cell or the
forespore. The results showed that
wild-type SpoIIIE forms a focus
only in the mother cell, supporting
the second model discussed
above. However, analysis
of translocation-defective
SpoIIIE showed that,
in the absence of DNA
translocation, SpoIIIE
complexes formed on
both sides of the septum,
supporting the first
model.
To resolve this
conundrum, Becker and
Pogliano moved on to look
at the effects of chromosome
orientation. In a B. subtilis strain
in which the chromosome par-
titioning proteins Soj and Spo0J
had been deleted, the SpoIIIE
complex could assemble in, and
move DNA out of, the forespore.
Finally, using a range of time-lapse
fluorescence microscopy studies the
authors were able to show that there
was a direct correlation between the
polarity of SpoIIIE-mediated chro-
mosome segregation and the position
of oriC after septation — SpoIIIE
moves the chromosome into the cell
that contains the replication origin.
So, it seems that aspects of both
models of SpoIIIE/FtsK function
were correct — the assembly of a
stable SpoIIIE translocation complex
is indeed cell-specific, but this spe-
cificity, and the resulting transloca-
tion polarity, are determined by the
orientation of the chromosome.
Sheilagh Molloy
ORIGINAL RESEARCH PAPER Becker, E. C. &
Pogliano, K. Cell-specific SpoIIIE assembly and
DNA translocation polarity are dictated by
chromosome orientation. Mol. Microbiol. 66,
1066–1079 (2007)

V IR U S S T R U C T U RE
One of a kind!
Despite the availability of a
commercial vaccine, the highly
contagious measles virus remains
a substantial health risk for which
there is currently no specifically
targeted treatment or antiviral
therapy. The structure of the measles
virus haemagglutinin (MVH), which
the virus uses to bind to host-cell
receptors, has now been revealed in
a recent study published in Nature
Structural & Molecular Biology.
Uniquely, species of Morbillivirus
(such as the measles virus) lack
neuraminidase activity and, instead
of binding to the host using sialic
acid, these viruses bind directly to
the host-cell receptors SLAM and/or
CD46. In this study, Colf and col-
leagues solved the structure of MVH
at a resolution of 2.7 Å. The overall
fold was similar to that of a
β-propeller, with six interconnected
blades (B1–6) — each of which
contains four antiparallel β-strands
— that surround a large cavity. Four
potential N-linked glycosylation sites
were detected, and the authors were
able to model the glycan electron
densities of two of these, Asn200 and
Asn215, so supporting the theory
that asparagine-linked glycosylation
enables MVH folding and stablization.
The authors then compared
the structure of MVH with those
of other viruses and found that it
was most similar to the haemag-
glutinin/neuraminidase fold of the
parainfluenza virus (PIV). However,
nature reviews | microbiology volume 6 | january 2008
© 2008 Nature Publishing Group
Research highlights
B1
B2
N-linked
glycosylation
Asn215
B3
Asn200
B6
B4 B5
C
N
Cartoon of the structure of the measles virus haemagglutinin. Figure 1b from Colf, L. A., Juo, Z. S. & Garcia,
K. C. Structure of the measles virus hemagglutinin. Nature Struct. Mol. Biol. 18 Nov 2007 (doi:10.1038/
nsmb1342).
large positional differences were neuraminidase structural fold that
detected, which suggests that, despite is similar to other viruses, such as
having similar folds, the haemag- PIV, but share few other structural
glutinin structures of the two viruses or sequence similarities. By using
have diverged considerably. Finally, the structure of MVH as a template,
mutagenesis and antibody-blocking structure-based drugs could be
data were mapped onto the solved designed that target the measles virus
MVH structure and it was found that at its point of entry and bring us one
the binding surfaces of SLAM and step closer to treating this potentially
CD46 are far apart and distinct from fatal viral disease.
the large cavity that is analogous to Gillian Young
that used by other viruses for sialic
acid binding. Original Research Paper Colf, L. A., Juo, Z. S.
This study further demonstrates & Garcia, K. C. Structure of the measles virus
the unique features of species hemagglutinin. Nature Struct. Mol. Biol. 18 Nov 2007
(doi:10.1038/nsmb1342)
of Morbillivirus, which retain a

b ac t erial v ir u lence
The cycle of success for Legionella
Two recent papers have provided
an insight into the mechanisms that
Legionella pneumophila uses to sub-
vert the host-cell vesicular trafficking
pathway to create its replicative niche.
L. pneumophila must establish
a replicative niche within alveolar
macrophages. This process involves
remodelling of the Legionella-
containing vacuole (LCV) by exploit-
ing the ability of the host protein
Rab1 to recruit material from the
vesicular transport pathway between
the endoplasmic reticulum and Golgi
apparatus. Rab proteins achieve this
by reversibly associating with lipid
membranes through a process that
is known as Rab membrane cycling.
Active GTP-bound prenylated Rab
proteins are present in membranes.
Once inactivated by a GTPase-
activating protein (GAP), GDP–Rabs
are removed from the membrane and
maintained in an inactive form in
the cytosol through association with
a guanine nucleotide-dissociation
inhibitor (GDI). The GDI can be
displaced by a GDI-displacement
factor (GDF), and the GDP-bound
nature reviews | microbiology volume 6 | january 2008
© 2008 Nature Publishing Group
Research highlights
Rab protein is then recruited back
to the membrane and activated by a
guanine nucleotide-exchange factor
(GEF). After the active Rab has
carried out its function, membrane
cycling is completed when the Rab is
inactivated by a GAP protein.
L. pneumophila uses a type IV
secretion system to secrete effector
proteins into the host-cell cytoplasm.
Previous work had shown that one
type IV effector, DrrA/SidM, is
required for the recruitment of Rab1
to the LCV and has Rab1-specific
GEF activity. Machner and Isberg,
and Ingmundson et al. were inter-
ested in whether DrrA/SidM also has
GDF activity. Both groups purified
overexpressed, tagged forms of Rab1
and Rab-GDI from eukaryotic cells to
ensure that they were prenylated and
then showed that DrrA/SidM inter-
feres with Rab1–Rab-GDI complex
formation by displacing the Rab-GDI,
thus confirming that DrrA/SidM
does have GDF activity. Machner
and Isberg went on to look at the
effects of the presence of liposomal
membranes. They found that the
activation of Rab1 by the GEF activ-
ity of DrrA/SidM was enhanced in
the presence of a lipid bilayer. They
were then able to follow the associa-
tion of the proteins with the mem-
brane during nucleotide exchange,
which confirmed that once the GDF
activity of DrrA/SidM has displaced
the Rab-GDI, the free Rab1 protein
is inserted in the membrane and can
then be activated by the GEF activity
of DrrA/SidM.
Both groups were also interested
in other L. pneumophila type
IV effectors that might function
downstream of DrrA. Machner and
Isberg showed that L.  pneumophila
LidA binds GTP–Rab1 that has been
activated by DrrA/SidM and sup-
ports the accumulation of activated
Rab1 on the LCV. Ingmundson et
al. found that LepB is delivered into
the host-cell cytoplasm shortly after
bacterial uptake, is present on the
early LCV membrane and can dis-
rupt secretory transport in a mam-
malian cell line. They completed
their work on LepB by demonstrat-
ing that this L. pneumophila effector
has GAP activity towards Rab1 and
propose that this activity completes
the membrane cycling of Rab1 that
is begun by DrrA/SidM.
DrrA/SidM is the first protein to
be identified that has both GDF and
GEF activity. Both groups point out
that this raises the possibility that
there might be eukaryotic GEFs
that have similar dual functions.
Sheilagh Molloy
ORIGINAL RESEARCH PAPERS Machner, M. P.
& Isberg, R. I. A bifunctional bacterial protein
links GDI displacement to Rab1 activation.
Science 318, 974–977 (2007) | Ingmundson, A. et al.
Legionella pneumophila proteins that regulate
Rab1 membrane cycling. Nature 450, 365–369
(2007)
news & analysis
genome watch
A poultry existence
Helena Seth-Smith
The two poultry pathogens discussed in
this month’s Genome Watch are closely
related to well-characterized organisms
that infect humans, so scrutinizing
their genomes could reveal factors that
determine host-specificity.
Avian influenza has the potential to ravage
the poultry industry and be transmitted to
humans. Bacterial pathogens of poultry are
also economically important. Bordetella avium
causes non-lethal respiratory disease, which
can predispose birds (mainly turkeys) to
secondary infections. Infection
of the respiratory tract by
avian pathogenic Escherichia
coli (APEC) strains can result
in colibacillosis, which can be
lethal.
The genome of B. avium
consists of 3,732,255 base
pairs and contains 3,417
coding sequences (CDSs)1.
The genome of the related
strain Bordetella bronchisep-
tica, which is responsible
for respiratory disease in
mammals, was sequenced
previously. A comparison of
the two could pinpoint those
genomic features that are
important to host specifi-
city, that is, the infection of
either an avian or a mamma-
lian host. These 2 strains share a
conserved Bordetella backbone
of 2,380 orthologous CDSs that
constitutes two thirds of the
B. avium genome. The CDSs that
are unique to B. avium mainly
encode surface-associated pro-
teins that might be important
in adhesion and immune-system
evasion. The B. avium genome
 | january 2008 | volume 6 www.nature.com/reviews/micro
contains CDSs that encode many novel
adhesins that are presumably specific for the
avian trachea, including several filamentous
haemagglutinins. There are potentially novel
biosynthetic pathways for both lipopolysac-
charide (LPS) and capsular polysaccharide,
which would also modify the bacterial-cell
surface. There are eleven CDSs that seem
to encode fimbriae, which could aid bind-
ing of the bacteria to cells of the respiratory
tract. The interaction with the host might
also be mediated by 2 novel surface proteins,
each of which comprises more than 4,000
amino acids, and 7 autotransport-
ers. Bordetella species often syn-
thesize toxins; B. avium can
produce dermo­necrotic toxin,
which has been shown to
be important in causing
disease in turkeys, and
tracheal cytotoxin.
Most E. coli strains are
associated with the intes-
tine. Strains that have
strayed from this lifestyle are
known as ExPEC (extraintes-
tinal pathogenic E. coli) and include
APEC and UPEC (uropathogenic)
strains, of which the UPEC strains are
responsible for urinary-tract infec-
tions in humans. Multiple E. coli
genomes have been sequenced,
including laboratory strains, those
that cause food poisoning and
other ExPEC strains. It has been pro-
posed that UPEC originated from
APEC, and genome comparisons
could help us to understand these
evolutionary connections.
Strain APEC 01 has a chromosome
of 5,082,025 base pairs, and comprises
4,467 CDSs2. It also harbours four
plasmids: one involved in virulence;
one involved in antibiotic resistance;
© 2008 Nature Publishing Group
and two cryptic plasmids. Comparative anal-
yses revealed that 78% of the CDSs from the
APEC 01 chromosome are common to all
sequenced E. coli strains, which means that
these CDSs comprise the minimal E. coli
backbone. A further 9% of the CDSs are
shared among all the sequenced ExPECs,
which might indicate their importance to the
survival of the bacteria outside the intestine.
These genes include those that code for LPS
synthesis, fimbriae, virulence‑associated pili
and iron acquisition. The remaining 13% of
CDSs are unique to APEC 01 and are pre-
dominantly located on 4 APEC‑specific
pathogenicity islands (PAIs), which are
phage-related and might contain additional
virulence factors.
The differences between the avian and
mammalian Bordetella strains seem to
reflect evolution over millions of years
through the vertical transfer of genes. The
E. coli genomes show more evidence of hori-
zontal gene transfer, and many of the differ-
ences between strains are restricted to PAIs
and plasmids.
Helena Seth-Smith is at the Sanger Institute,
Wellcome Trust Genome Campus, Hinxton,
Cambridge CB10 1SA, UK.
e-mail: microbes@sanger.ac.uk
doi:10.1038/nrmicro1830
1. Sebaihia, M. et al. Comparison of the genome sequence
of the poultry pathogen Bordetella avium with those of
B. bronchiseptica, B. pertussis, and B. parapertussis
reveals extensive diversity in surface structures
associated with host interaction. J. Bacteriol. 188,
6002–6015 (2006).
2. Johnson, T. J. et al. The genome sequence of avian
pathogenic Escherichia coli strain O1:K1:H7 shares
strong similarities with human extraintestinal
pathogenic E. coli genomes. J. Bacteriol. 189,
3228–3236 (2007).
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
APEC 01 | Bordetella avium | Bordetella bronchiseptica
All links are active in the online pdf
 disease watch | in the news
Choose your battles wisely
Neil Smith
Animals, like plants, can respond in
different ways to parasite infections: they
can mount a full-scale immune response
or they can tolerate the invader. Andrew
Read and colleagues from the University
of Edinburgh, UK, introduced rodent
malaria into five strains of laboratory mice
and monitored parasite load and animal
health, as measured by anaemia and body
mass. In some strains of mice, parasite load
increased and the mice stayed healthy,
which indicated that the mice could
tolerate the parasite. In other mice strains,
however, parasite load decreased, which
indicated that the mice were resisting
infection. The authors suggest that these
findings could have important implications
for pathogen evolution: pathogens might
not be pushed to evolve increased virulence
if they are tolerated rather than destroyed.
However, tolerated pathogens are also
more likely to spread.
In a somewhat related paper, Lynn Martin,
from the University of South Florida, and
colleagues report that fast-living strains
of mice develop high fevers in response to
simulated infection, but slow-living strains
do not. The authors propose that the ‘live
fast, die young’ mice tolerate the harm
that fever inflicts on bodily tissues, as they
already have short lifespans. Slow-living
mice, however, have more to lose and may
therefore adopt targeted strategies, such
as antibody production. Science/Funct. Ecol.
nature reviews | microbiology volume 6 | january 2008 | 
Dandruff on the collar?
The genome of Malassezia globosa — a fungus
that causes discomforting and embarrassing
dandruff — has been sequenced by an
international team led by Thomas Dawson
at Procter & Gamble. Malassezia spp.-
associated dandruff affects more than 50%
of humans, and more than UK£3 billion are
spent annually on dandruff treatments.
Genome sequencing revealed that the
M. globosa genome is among the smallest
of all the free-living fungi analysed so far,
containing as few as 4,285 predicted protein-
coding genes. Of these, approximately 50
encode secreted proteins, including many
lipid-degrading proteins, that are probably
responsible for breaking down hair and scalp,
thereby causing the irritation, inflammation
and flaking that are hallmarks of dandruff. Not
only might this sequenced genome represent
a gold mine for developers of anti-dandruff
shampoos, they also have agricultural
implications as M. globosa is closely related
to various common pathogenic plant fungi
that affect corn, wheat and other food crops.
PNAS/The Telegraph
Novel MRSA virulence factors
identified
Although community-acquired meticillin-
resistant Staphylococcus aureus (CA-MRSA)
causes the majority of the staphylococcal
infections that result in trips to emergency
rooms, the basic cause of CA-MRSA
virulence remains unidentified. However,
a class of secreted cytolytic peptides
(so‑called PSMs) that function primarily to
destroy leukocytes has now been identified
by Michael Otto, from the Rocky Mountain
Laboratories, USA, and colleagues. Deletion
of PSM genes resulted in a decrease in the
severity of CA-MRSA in two mouse abscess
and bacteraemia models. Moreover, in vitro
studies indicated that PSM genes caused
S. aureus to recruit, activate and lyse human
neutrophils. Notably, hospital-acquired
MRSA strains exhibit a significantly
lower expression of PSM genes than
do CA-MRSA strains, which are
more virulent. Nature Med.
Vaccine may have
increased HIV risk
In late September, a trial of Merck’s
candidate HIV vaccine, which included
over 3,000 people from 9 countries,
© 2008 Nature Publishing Group
was unexpectedly ended after preliminary
results showed that the vaccine was not
conferring resistance. Further analysis
suggests that the vaccine — which
consists of 3 HIV genes and an attenuated
adenovirus 5 vector — may have increased
HIV susceptibility in recipients. Notably,
21 of 392 male vaccine-recipient patients
with pre-existing immunity to adenovirus 5
acquired HIV infections, compared with
9 of 386 from the placebo group. As the
trial was halted abruptly, researchers
cannot determine whether these results
are statistically significant. Among
other explanations, vaccination may
have increased the production of CD4+
T cells — HIV’s favorite infection target
— in adenovirus-5-immune patients. An
upcoming trial of a vaccine developed by
the National Institutes of Health has been
delayed to allow further analysis. Nature/
New York Times
HIV numbers revised
The United Nations has lowered their
estimate of how many people are infected
with HIV globally from 39.5 million to
33.2 million. These revised numbers,
however, are attributed to improved
methods of data collection rather than to
a real decline in HIV infection. The number
of HIV carriers is still rising, although
the global prevalence of HIV seems to
be levelling off. An estimated 2.5 million
new infections occurred in the past year
(which works out at a staggering 6,800 new
infections per day), down from a likely peak
in the late 1990s of over 3 million infections
per year. Sub-Saharan Africa remains the
hardest hit by HIV, accounting for nearly
50% of the new cases of infection and 68%
of the total number of cases. WHO
STOCKBYTE
N e w s & a n a ly s i s
Use condoms!
Although the global prevalence of HIV
is showing signs of levelling off, the
transmission rates for sexually transmitted
infections (STIs) in the United Kingdom
are still on the rise, despite concerted
public health efforts to reverse this trend.
During 2006, report the Health Protection
Agency (HPA), 376,508 STIs were newly
diagnosed — a 2.2% increase compared
with the number of new STIs in 2005. The
number of STIs diagnosed each year in
the United Kingdom has increased almost
continually since the 1990s. In particular,
the sexual health of young adults has
worsened, although the HPA also warns of
a continuing HIV and STI epidemic among
men who have sex with men. An estimated
73,000 adults in the United Kingdom are
now HIV-positive, although one-third of
these individuals remain unaware that they
are infected. More funding and education
is needed to tackle these problems, say
experts. HPA/BBC
Can Africa cope with avian
influenza?
African nations are unlikely to be able to
achieve the five priorities that have been
identified by the World Health Organization
(WHO) to fight avian influenza, argue
Robert Webster and colleagues. So far there
have been only 40 cases of avian influenza
in Africa, for which there has been a 40%
fatality rate. However, the predominant
strain of avian influenza in Africa, the
Qinghai strain, “has acquired several
troubling properties, including respiratory
rather than faecal transmission in poultry.”
The five priorities of the WHO for the
combat of avian flu are: reduce human
exposure; strengthen surveillance; intensify
rapid containment; enhance response
capacity; and coordinate global research.
Owing to a number of factors, including
inadequate surveillance, the denial of
outbreaks and the poor communication
of risks to the public, African nations are
unlikely to achieve these targets in the
opinion of some observers. For example,
it took 4 weeks to officially confirm the
outbreaks in all of the affected African
countries, apart from Egypt. Webster and
colleagues call for each African nation
to realistically assess its status, conduct
regular active surveillance and be more
forthcoming with data. Lancet Infect. Dis.
10 | january 2008 | volume 6 www.nature.com/reviews/micro
Anti-polio programme gets
a booster
IMAGE SOURCE
The campaign to eradicate polio has
received a much-needed US$200 million
(£97 million) grant from the Bill and Melinda
Gates Foundation and Rotary International.
An initial $100 million will be distributed
through grants to the WHO and UNICEF to
support mass immunization campaigns in
polio-affected countries, polio-surveillance
activities, and community-education
and outreach programmes. Although
immunization programmes have made
huge strides over the past 20 years, polio is
still endemic in Nigeria, Pakistan, India and
Afghanistan, and approximately 700 cases
of polio are reported each year. Despite this
generous donation, WHO officials report
that the polio-eradication programme is still
facing a $650 million shortfall over the next
2 years. BBC
Cracking cholera’s lines of
communications
The Vibrio cholerae signalling molecule
cholera autoinducer-1 (CAI-1) may form
the basis of therapeutic intervention
against V. cholerae infection, reports
Bonnie Bassler and colleagues. V.
cholerae, and other bacteria, use quorum
sensing (QS) to sense and respond to
population density, and this cell-to-
cell communication is mediated by
© 2008 Nature Publishing Group
autoinducers. V. cholerae produces
two autoinducers, autoinducer 2 (AI‑2)
and CAI‑1. The structure of AI-2 has
been known for many years, and in this
latest paper Bassler et al. characterized
and synthesized CAI‑1. To assess the
feasibility of controlling V. cholerae by
manipulating QS the authors showed that
synthetic CAI‑1 can repress production
of the V. cholerae virulence factor toxin
co-regulated pilus as well as natural CAI-1.
“This work”, the authors argue, “provides
a demonstration that interference with
quorum-sensing processes in general ... has
great promise in the clinical setting.” Nature
Outbreak news
Rift Valley Fever. An outbreak of Rift Valley
Fever has killed 164 people out of a total
of 451 who have contracted the disease
in Sudan. The majority of these infections
are the result of direct or indirect contact
with the blood and organs of infected
animals, although some cases are also the
result of bites from infected mosquitoes.
Sudanese health authorities initially
sought help to control the outbreak in
mid-October. Rift Valley Fever normally
has a fatality rate of ~1%, but the fatality
rate is ~50% in patients who develop the
haemorrhagic form. WHO/Associated Press
Encephalitis. A ‘mystery’ pathogen
that causes encephalitis has resulted in
21 deaths, mostly children, in a remote
region of Bangladesh. An additional
200 people are affected. Bangladesh’s
International Epidemiology Research
Centre and the Centers for Disease
Control and Prevention in the United
States are working together to identify the
causative pathogen. Reuters
Ebola. WHO officials have confirmed
that Ebola virus has killed 16 people and
affected an additional 50 in western
Uganda. The virus belongs to a distinct
subtype that has not been detected before,
as indicated through tests conducted by
national laboratories in Uganda and the US
Center for Disease Control. Ebola is fatal in
approximately 80% of cases, and as yet there
is no known cure. BBC
In the News was compiled with the assistance of
David Ojcius, University of California, Merced,
USA. David’s links to infectious disease news
stories can be accessed on Connotea (http://www.
connotea.org), under the username ojcius.
 Progress
promoting bacterial basolateral entry into

The versatility of Shigella effectors polarized epithelial cells1,4 (FIG. 1).

IpaA binds the amino-terminal head
domain of vinculin, which is a key com-
Michinaga Ogawa, Yutaka Handa, Hiroshi Ashida, Masato Suzuki and ponent of focal adhesions, and stimulates
actin depolymerization5. IpaA also targets
Chihiro Sasakawa
β1-integrin and stimulates the GTPase
Abstract | When Shigella infect the intestinal epithelium, they deliver several activity of RhoA, thereby inducing the loss
of actin stress fibres6. Thus, IpaA might
effectors through the type III secretion system (T3SS) into the surrounding space
facilitate recycling of the free actin pool
and directly into the host-cell cytoplasm, where they can mimic and usurp host by the destruction of stress fibres and
cellular functions or subvert host-cell signalling pathways and the immune contribute to the production of membrane
response. Although bacterial strategies and mechanisms of infection vary greatly, ruffles. The interaction between IpaB and
recent studies of Shigella effectors have revealed that Shigella possess a highly the CD44 receptor, which is present on the
evolved strategy for infection. epithelial cell membrane in areas that are
rich in lipid rafts, stimulates basolateral
invasion7. IpaB is a multifunctional effec-
The intestinal epithelium acts as an intrinsic which eventually leads to entry by macro­ tor2,4,8. As well as being secreted at the tip
defensive barrier against microbial invaders. pinocytosis. As soon as a bacterium is of the T3SS needle and acting as a T3SS
The barrier function has four major ele- surrounded by a membrane vacuole within translocator (with IpaC), it is also involved
ments: the resident commensal bacteria; an epithelial cell, it disrupts the vacuolar in vacuole disruption, activates caspase‑1
the integrity of the epithelial barrier; membrane and escapes into the cytoplasm. in macrophages and modulates cell-cycle
the rapid epithelial-cell turnover; and the Shigella can multiply in the epithelial-cell progression in epithelial progenitor cells
innate immune response. Nevertheless, cytoplasm and move both intra- and inter- (discussed below). IpaC is delivered into
many pathogenic bacteria can circumvent cellularly, and so infection of the intestinal the epithelial-cell cytoplasm and integrates
these defences and exploit the intestine epithelium by Shigella elicits a strong into the membrane, where it is assumed
as a replicative niche, a shelter from host inflammatory response1,2. to have a key role, either directly or indi-
immune surveillance or a port of entry for To proceed through the series of steps rectly, in inducing actin polymerization4.
dissemination into deeper tissues. that are involved in infection, Shigella Although there is no direct evidence as
Shigella species (abbreviated here to secrete many effector proteins through the yet, IpaC might be involved in promot-
Shigella) are human-adapted pathogens that type III secretion system (T3SS)3. TABLE 1 ing Shigella invasion of epithelial cells by
are capable of colonizing the intestinal epi- lists some of the characterized Shigella inducing actin foci through the accumula-
thelium by exploiting epithelial-cell func- effectors and their homologues in other tion of the host non-receptor tyrosine
tions and circumventing the host innate bacterial pathogens. This Progress article kinase Src at the site of bacterial entry4. On
immune response. Shigella have neither highlights recent advances in our under- entry, the phosphorylation of cortactin — a
adherence factors nor flagella. Following standing of the roles of the Shigella effec- cortical actin-binding protein that activates
ingestion by the faecal–oral route, Shigella tors that are delivered into host cells by the the Arp2/3 complex and induces actin
move through the small intestine to the T3SS during intestinal infection. polymerization — occurs in a Src-dependent
colon and rectum, where they cross the epi- manner and Crk (a Src-related tyrosine
thelial barrier through microfold cells that Effectors in the early stages of infection kinase) activates the small GTPase Rac1
overlie solitary lymphoid nodules. When The ability to invade, colonize and trans- (Ref. 4).
the epithelial barrier has been breached, locate across mucosal barriers is an impor- IpgB1 is assumed to have a major
Shigella immediately enter the macrophages tant feature of enteric pathogens. Notably, role in producing membrane ruffles by
that reside within the microfold‑cell pocket. in the case of Shigella, the ability to invade activating Rac1 through ELMO–Dock180,
Once within the macrophages, the infecting epithelial cells and subsequently spread a Rac1 guanine nucleotide-exchange
bacteria disrupt the phagosomal membrane from cell to cell is pivotal in establishing factor9. For example, when IpgB1 is
and disseminate from the phagosome into an intestinal infection. When Shigella ectopically expressed in HeLa cells, large
the macrophage cytoplasm, where they come into contact with epithelial cells, membrane ruffles are produced. Pull-
multiply and induce rapid cell death1,2. they deliver a subset of effectors through down assays have identified ELMO as
Shigella that are released from dead the T3SS both around the bacterial surface the IpgB1-binding partner; IpgB1 binds
macrophages can enter the surrounding and directly into host cells. These effectors, to the amino‑terminal region of ELMO,
enterocytes by inducing the production of which include IpaA, IpaB, IpaC, IpgB1, which also binds RhoG. An in vitro bind-
membrane ruffles at the basolateral surface, IpgB2, IpgD and VirA, are involved in ing assay showed that RhoG binding to

nature reviews | microbiology volume 6 | january 2008 | 11

© 2008 Nature Publishing Group
Progress

Table 1 | Activities of Shigella type III secretion system (T3SS) effectors

T3SS Biochemical Target (or targets) Role in infection Selected homologues Refs
effector activity
IpaA Unknown Vinculin Bacterial invasion Salmonella spp. SipA (also called 9,10
SspA)
IpaB T3SS translocon Caspase-1, CD44 Macrophage apoptosis and Salmonella spp. SipB (also called SspB) 6,34,36
and Mad2L2 cell-cycle arrest and Yersinia spp. YopB
IpaC T3SS translocon Unknown Bacterial invasion Salmonella spp. SipC (also called SspC) 37
IpgB1 RhoG mimic ELMO Bacterial invasion Salmonella spp. SifA and SifB; EHEC, 11,12
EPEC and Citrobacter rodentium Map;
and EHEC EspM1 and EspM2
IpgB2 RhoA mimic mDia and ROCK Unknown
IpgD Inositol phosphate Phosphatidylinositol Bacterial invasion and host-cell Salmonella spp. SopB (also called 7,8
phosphatase 4,5-bisphosphate survival SigD)

VirA Cysteine protease Tubulin Bacterial invasion and intracellular EHEC, EPEC and C. rodentium EspG 13,14,17
spreading and EPEC EspG2 (also called Orf3)

IcsB Unknown Shigella VirG (also Escape from autophagy Burkholderia spp. BopA 20
called IcsA)
OspC1 Unknown Unknown Polymorphonuclear transepithelial Shigella OspC2, OspC3 and OspC4 38
migration and Vibrio parahaemolyticus OspC2
OspE2 Unknown Unknown Intercellular spreading Shigella OspE1; Salmonella spp. 39
EspO1STYM; and EHEC EspO1-1 and
EspO1-2
OspF Phosphothreonine MAP kinases Suppression of innate immune Salmonella spp. SpvC; 27,29,38
lyase responses Pseudomonas syringae HopAI1; and
Chromobacterium violaceum VirA
OspG Serine/threonine E2 ubiquitin- Suppression of innate immune Yersinia enterocolitica YE2447; 26
kinase conjugating responses C. rodentium NleH; and EHEC NleH1-1
enzymes and NleH1-2
IpaH9.8 E3 ubiquitin ligase U2AF35 and yeast Suppression of innate immune Shigella IpaH4.5; Salmonella spp. 24,25
Ste7 responses SspH1, SspH2 and SlrP; Yersinia
pestis YP3416 and YP3418; P. syringae
IpaH7.8 E3 ubiquitin ligase Unknown Escape from endocytic vacuoles of PSPTO1492 and PSPTO4093; and 25,40
phagocyte Rhizobium spp. Y4fR
Chromosomal Possibly E3 Unknown Suppression of innate immune 23
IpaHs ubiquitin ligase responses
EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic E. coli, MAP, mitogen-activated protein.

ELMO is competitively inhibited by IpgB1,
implying that IpgB1 mimics the function
of RhoG in producing membrane ruffles
during Shigella invasion9. IpgB2 is an
IpgB1 homologue that binds to mDia1,
which facilitates actin nucleation, and the
Rho kinase ROCK through its GTPase-
binding domains. In this way, IpgB2
mimics the activity of RhoA in inducing
the formation of stress fibres, although
its specific involvement in bacterial inva-
sion remains unclear10. IpgD possesses
phosphatidylinositol (4,5) bisphosphate
(PI(4,5)P2) phosphatase activity and
catalyses the hydrolysis of PI(4,5)P2 to
phosphatidylinositol 5‑monophosphate
(PI(5)P), thereby promoting local actin
polymerization4,11,12.
VirA, which is a member of the EspG/
VirA family, shares significant amino-acid
homology, as well as functional similarity,
with EspG. EspG/VirA family members
are found in enteropathogenic Escherichia
coli (EPEC), enterohaemorrhagic E. coli
(EHEC) and Shigella. Intriguingly, VirA
is delivered into the host-cell cytoplasm
near the site of bacterial entry and induces
local microtubule (MT) degradation13. As
degradation of MTs by EspG results in the
release of various MT‑associated proteins,
including GEF‑H1, and GEF‑H1 activates
RhoA14, VirA activity is assumed to con-
tribute to ruffle formation during Shigella
invasion through the cross-talk between
RhoA and Rac1.
Clearly, these studies indicate that
Shigella exploit the interactions between a
subset of effector proteins and their host
target molecules (or target functions). Such
interactions are capable of stimulating sev-
eral host-cell signalling pathways that are
involved in inducing actin polymerization
and remodelling the architecture of the
host-cell surface (FIG. 1).
Intra- and intercellular motility
Many pathogenic bacteria can invade host
cells, and some species, such as Shigella,
Listeria monocytogenes, Mycobacterium
marinum, Rickettsia conorii and
Burkholderia pseudomallei, are capable of
inducing actin nucleation at one pole of the
bacterium to gain the propulsive force that is
necessary to move through the host cell and
into neighbouring host cells. This activity,
often called actin-based bacterial motility,
is crucial for establishing an infectious foot-
hold as well as renewing replicative niches.
Intriguingly, the bacterial proteins that are
involved in mediating actin nucleation vary
from species to species. Nevertheless, these
proteins share the ability to recruit and acti-
vate the Arp2/3 complex, which is required
for actin polymerization near the bacterial
surface8. In the case of Shigella, an outer-
membrane protein called VirG (also known
as IcsA) has a key role, as VirG can directly

12 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group
 Progress

cysteine-protease-like activity17. Consistent

with this observation, a virA mutant that
lacked this activity was found to be less
T3SS capable of moving smoothly within the host
RhoG ELMO IpgB1 Shigella cytoplasm than the wild type and was inca-
T3SS pable of maintaining continuous cell–cell
effectors spreading17.
Although there is no direct evidence
CD44 as yet, the ability to clear a path through
Dock180 IpaB
IpaC the host-cell MTs might not be unique to
IpgB2 Shigella. L. monocytogenes ActA is capable
mDia
IpgB2 of directly recruiting the Arp2/3 complex in
Rock the vicinity of the bacterial surface, and the
RhoA Rac1 Cdc42 destruction of MTs is occasionally detected
along the path of L. monocytogenes move-
ment. Intriguingly, L. monocytogenes ActA
is not a VirA homologue but it indirectly
WAVE IpaC recruits Op18, an MT‑sequestering host
Microtubule protein, near the bacterial surface18. It is
destabilization Major actin tempting to speculate that Op18 facilitates
rearrangements L. monocytogenes movement within epithe-
Arp2/3 Membrane lial cells. The discovery of a novel bacterial
PI(4,5)P2 ruffling activity that destroys MTs demonstrates that,
VirA
although MTs are an obstacle to bacterial
IpgD movement within the host-cell cytoplasm,
IpaA Entry certain bacteria have evolved an activity to
IpaA
Vinculin remove this barrier.

Escape from autophagy

IpaB
α5β1 integrin Autophagy is a ubiquitous degradation
IpaC
system in eukaryotic cells that is required
not only for the cellular response to starva-
tion and stress, and for the removal of
damaged or surplus organelles, but also
T3SS effectors for removing bacterial pathogens that
Figure 1 | A simplified model of membrane-ruffle production in response to the stimulation invade the cytoplasm of host cells. For
of host cellular signalling by Shigella effectors. Upon contact between Shigella and an epi- example, Streptococcus pyogenes (Group A
thelial cell membrane, the bacterium delivers several effectors through the type III secretion Streptococcus) and Staphylococcus aureus are
Nature Reviews
system (T3SS) around the bacterial surface and into the host-cell cytoplasm. | Microbiology
By interacting with capable of invading epithelial cells, and both
their host binding partners the effectors are eventually able to activate the Rac1–WAVE–Arp2/3 pathogens are targeted by the autophagic
pathway, which leads to the protrusion of membrane ruffles around the bacterial entry point. It
machinery and eventually undergo lyso-
should be noted that this model does not rule out any other additional, as yet uncharacterized,
somal degradation. Autophagy is achieved
signalling pathways that may be involved in mediating actin polymerization during Shigella
invasion. See the main text for details. by a series of autophagy-related (Atg)
proteins that are highly conserved from
yeast to humans19. During multiplication
within epithelial cells, Shigella are recognized
interact with N‑WASP (neural Wiskott– The movement of bacteria within host by components of the autophagic pathway.
Aldrich syndrome protein)15. When VirG cells is highly variable and depends on their However, the secretion of IcsB through the
and Cdc42 interact with N‑WASP, N‑WASP location in the host cell. For example, some T3SS on entry into the cytoplasm allows the
becomes activated, which, in turn, recruits motile Shigella suddenly change direction, bacteria to escape autophagic destruction20.
and activates the Arp2/3 complex16. The spin around or stop moving. It has recently Although an icsB mutant is fully invasive
formation of the VirG–N-WASP–Arp2/3 been shown that Shigella movement within and can escape from the phagocytic vacuole
complex at one pole of the bacterium allows the host-cell cytoplasm is severely hindered in epithelial cells, it is ultimately enclosed
Shigella to induce actin nucleation and by MTs, but a motile bacterium can destroy by autophagosomes. Surprisingly, IcsB does
elongation, thus gaining propulsive force surrounding MTs using VirA17 (FIG. 2). not directly inhibit autophagy itself. Instead,
(FIG. 2). During bacterial movement, some As described above, VirA is also used to the VirG protein — which is required for
bacteria impinge on the host-cell membrane promote bacterial entry. Thus, VirA has dual actin-based bacterial motility (FIG. 2) — is
and cause the membrane to protrude and roles in both bacterial invasion and intracel- targeted for autophagic recognition by bind-
penetrate into that of neighbouring cells, lular spreading. Characterization of VirA ing to Atg5 (a protein that is involved in
thereby allowing the bacteria to disseminate activity indicated that the degradation of MTs the elongation of isolation membranes).
into adjacent cells. by VirA depends on its α‑tubulin-specific In in vitro binding assays, both IcsB and

nature reviews | microbiology volume 6 | january 2008 | 13

© 2008 Nature Publishing Group
Progress
Capping
protein Profilin
F-actin
Vinculin
Arp2/3
N-WASP
G-actin
Cdc42
Figure 2 | Shigella movement within the host-cell cytoplasm requires actin polymerization
and microtubule degradation. Asymmetric distribution of VirG Nature
bacterial surface is essential for the polar movement of Shigella in epithelial cells. The accumu-
lation of VirG at one pole of the bacterium recruits and activates the Arp2/3 complex by the
interaction between VirG and N‑WASP (neural Wiskott–Aldrich syndrome protein). Motile
Shigella secrete VirA through the type III secretion system and destroy local microtubules (MTs),
thereby facilitating bacterial movement.
Atg5 can bind to VirG. Intriguingly, IcsB
and Atg5 share the same binding region on
VirG, and the affinity of IcsB for VirG is
much stronger than the affinity of Atg5 for
VirG, which suggests that IcsB acts as an
anti-Atg5-binding protein. The interaction
between IcsB and VirG near the bacte-
rial surface, therefore, seems to provide
a mechanism of escape from autophagic
recognition1,20. L. monocytogenes can also
escape autophagy during multiplication in
the host-cell cytoplasm, but the mechanism
that underlies this escape seems to differ
from that of Shigella21. Although the precise
mechanism is still unknown, in contrast
to Shigella VirG, ActA, which is present on
the surface of L. monocytogenes and medi-
ates actin polymerization at one pole of
the bacterium, seems to be involved in the
escape of L. monocytogenes from autophagic
recognition.
The recognition of intracellular pathogens
by autophagy is not limited to pathogens
that invade the cytoplasm. As long as the
innate immune response of the host is
intact, intracellular pathogens, such as
Mycobacterium tuberculosis, Legionella pneu-
mophila and Coxiella burnetii, which are
sequestered in vacuolar compartments, can
be also targeted by autophagy. Unless they
are able to modify the vesicles in which
they are contained or avoid autophagic
uptake at an early stage of infection, they
are entrapped by a lamellar membranous
structure that is associated with LC3, an
essential autophagic protein. Although
the mechanisms that are involved remain
14 | january 2008 | volume 6 www.nature.com/reviews/micro
MT network
VirA
VirA
IcsB
VirG Shigella
VirA
IcsB
VirA
VirA
(also known
Reviewsas| Microbiology
IcsA) on the
unclear, recent studies have indicated that
these intracellular pathogens are also capa-
ble of certain manoeuvres that allow them
to circumvent autophagic recognition.
Modulation of the innate immune response
Major bacterial-cell components, such as
lipopolysaccharide (LPS), peptidoglycan
(PGN), and, perhaps, nucleic acids (DNA
and RNA), are readily released from
multiplying and killed bacteria. These
components are recognized by Toll-like
receptors and Nod-like receptors, which
activate the host innate defence systems,
stimulate inflammatory signalling cascades
and induce cellular and humoral immune
responses. Nevertheless, many bacterial
pathogens that infect the intestinal mucosa
can circumvent the host innate immune
response and colonize their replicative
niches2. The mechanisms that are used by
bacteria to dampen host inflammatory
signals have been extensively studied using a
range of animal and plant pathogens.
Shigella release LPS and PGN into host
cells, and these pathogen-associated molecular
patterns are the main cause of the strong
inflammatory response that is induced by
Shigella infection2. For example, the LPS that
is released by Shigella as they escape from
phagosomes into the macrophage cytoplasm
activates caspase‑1, which induces the produc-
tion of interleukin‑1β and cell death. PGN
released from Shigella that are multiplying
within epithelial cells activates the Nod1–RICK
pathway, which stimulates signalling cascades
that involve mitogen-activated protein kinases
© 2008 Nature Publishing Group
(MAPKs) and nuclear factor (NF)-κB and
results in the production of proinflammatory
cytokines22.
To circumvent the innate immune
response, Shigella deliver more than ten
effector proteins, including the IpaH
proteins, OspG and OspF, into host cells
through the T3SS2,23 (FIG. 3; TABLE 1).
IpaH9.8, a member of the IpaH protein
family, translocates to the nucleus, where
it interacts with U2AF35, an mRNA splic-
ing factor24. Infection of epithelial cells
by an ipaH9.8 mutant increased the levels
of proinflammatory cytokines, and RNA
interference (RNAi)-mediated knockdown
of U2AF35 production decreased these
levels. In a murine lung-infection model,
the ipaH9.8 mutant induced more severe
inflammatory responses and greater
proinflammatory cytokine production than
did wild-type Shigella, which resulted in a
30-fold decrease in bacterial colonization24.
It has recently been suggested that the IpaH
homologues (including IpaH9.8) that are
produced by plant and animal pathogens,
such as Yersinia species, Salmonella spe-
cies, Edwardsiella ictaluri, Bradyrhizobium
japonicum, Rhizobium species and some
Pseudomonas species, share an E3 ubiquitin
ligase activity in the highly conserved
carboxy‑terminal region25 (TABLE 1).
Although the host target (or targets) of each
ubiquitin ligase remains unknown, in a
yeast-cell system, IpaH9.8 was shown to have
an activity that interferes with the pherom-
one response by the ubiquitination of the
MAPK kinase Ste7, which is then degraded
by proteasomes25. These findings suggest
that IpaH and IpaH homologues have a
central role in dampening host inflam-
matory-related signals during bacterial
infection (FIG. 3).
OspG can bind to ubiquitinated E2s
(ubiquitin-conjugating enzymes), such as
UbcH5b, a component of the Skp1–culin–F-
box protein (SCF)β–TrCP complex that is
involved in phospho‑IκBα ubiquitination
and its subsequent degradation by the
proteasome26. Thus, OspG interferes with
IκBα degradation, thereby resulting in a
repression of NF‑κB activation. Consistent
with this observation, the characterization of
the phenotype of an ospG mutant in in vitro
and in vivo models of infection has shown
that OspG is involved in downregulating
the host inflammatory response to Shigella
infection26.
OspF also translocates to the host-cell
nucleus. OspF has a specific phosphatase
activity that dephosphorylates and inactivates
MAPKs, such as ERK1/2, JNK and p38,

thereby blocking the phosphorylation of the
serine 10 residue of histone H3, which is
required for the transcription of a subset of
NF‑κB-regulated genes27–29. Intriguingly, in
contrast to the OspF activity that is proposed
above, Shigella OspF, Salmonella spp. SpvC
and Pseudomonas syringae HopAl1 have also
been shown to share the ability to dephos-
phorylate MAPKs through their phospho-
threonine lyase activities, thereby enabling
these bacterial effectors to interfere with
MAPK activity3,29 (TABLE 1). In rabbit ileal
loops and a mouse-lung infection model,
the ability of a Shigella ospF mutant to
induce inflammatory responses was much
greater than that of wild-type Shigella.
Although there is some controversy regard-
ing the activity of OspF, which might be
attributed to the different experimental
conditions that are used in each study or
the dual activities that are encoded by
OspF, these studies have clearly indicated
that OspF participates in dampening the
inflammatory response27.
The reason for the presence of such a vari-
able number of effectors in Shigella and other
pathogens remains a matter of speculation.
As numerous signal-transduction pathways
are engaged in inducing the inflammatory
response to bacterial infection, many effec-
tors must be engaged in modulating the
various inflammatory signal pathways at
different levels and different times during
bacterial infection.
Slowing of rapid epithelial-cell turnover
The intestinal epithelium renews itself
every several days, which provides an
important intrinsic defence system that
limits bacterial colonization. The rapid
turnover of intestinal epithelial cells forms
a crucial physical, as well as functional,
barrier, and renewal is sustained by
the vigorous proliferation of epithelial
progenitor cells that migrate upwards
from the bottom of the intestinal crypts.
Nevertheless, many pathogenic bacteria,
including Shigella, are capable of coloniz-
ing the intestinal epithelium. Recent stud-
ies have indicated that a growing family of
bacterial toxins, effectors and small com-
pounds, called cyclomodulins, are capable
of interfering with the eukaryotic cell
cycle30,31. Some of the cyclomodulins — for
example, the EPEC protein cycling inhibi-
tor factor (Cif) — inhibit host-cell mitosis
after transfer from the bacteria through
the T3SS. Cells that have been transformed
by Cif accumulate 4n DNA and re-initiate
DNA synthesis without dividing, which
results in cells that contain 8–16n DNA31.
nature reviews | microbiology volume 6 | january 2008 | 15
Progress
Shigella
Nod1/2 T3SS
effectors OspG
PGN
LPS
E2s
RICK Ub
UbcH5b IpaHs
β-TrCP
IκB
IKK p65 p50
MAPK Ub Ub
complex IκB Unknown
NF-κB
Proteasome
OspF P
P IpaH9.8 U2AF35 Interleukin-8
MAPK MAPK production
IL-8
Histone H3 p65 p50
Figure 3 | Shigella and the downregulation of the host inflammatory response. During the
multiplication of Shigella in epithelial cells, the bacteria shed components,Naturesuch as peptidoglycan
Reviews | Microbiology
(PGN) and lipopolysaccharide (LPS), into the cytoplasm. The recognition of PGN by Nod1 activates the
nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK)-dependent inflammatory signals.
Shigella delivers a set of effectors, including IpaH9.8, IpaHs (IpaH9.8 homologues), OspF and OspG, into
the host cell through the type III secretion system (T3SS), which enables them to circumvent the host
inflammatory response and inactivate the innate immune system. See the main text for details.
Other cyclomodulins, the cytolethal Shigella IpaB is involved in mediating
distending toxins (CDTs), are produced by the translocation of effectors through the
Shigella dysenteriae, Campylobacter jejuni, T3SS and also functions as an invasin that
E. coli and Salmonella enterica serovar interacts with CD44 (Refs 1,4,7). It has
Typhi (S. typhi). One of the CDTs that is been shown that IpaB is delivered into the
produced by C. jejuni possesses a deox- epithelial-cell cytoplasm by intracellular
yribonuclease‑I-like activity and causes bacteria and causes cell-cycle arrest by
limited DNA damage when it is delivered targeting Mad2L2, an anaphase-promoting
into the host-cell nucleus, which leads to complex/cyclosome (APC) inhibitor34.
the activation of ATM (a PI3 kinase) and Cell-cycle progression is stringently con-
eventually results in cell-cycle arrest32. trolled by cell-cycle-specific proteolysis,
Helicobacter pylori VacA, which induces which involves the ubiquitination of
cellular vacuolation in epithelial cells, is target proteins by two main types of E3
also capable of efficiently blocking the ligase complexes: the APC complex and
proliferation of T cells by inducing G1/S the SCF complex35. The APC complex is
cell-cycle arrest33. Although the biological a multisubunit complex that targets sub-
importance of each cyclomodulin and strates for degradation only during mitosis
its target host cells in bacterial infection and the G1 phase; it also targets mitotic
remains largely unclear, it is expected that cyclin A and cyclin B1, so allowing mitotic
some cell-cycle inhibitors will prolong the progression. Cyclin B1 ubiquitination
pathogen’s presence by interfering with the assays have shown that APC undergoes
rapid turnover of epithelial cells. unscheduled activation in response to
© 2008 Nature Publishing Group
Progress
IpaB interaction with Mad2L2. Indeed,
synchronized HeLa cells infected with
Shigella fail to accumulate APC substrates,
such as cyclin B1, Cdc20 and Plk1, which
causes cell-cycle arrest at the G2/M phase
in an IpaB–Mad2L2-dependent manner.
IpaB–Mad2L2-dependent cell-cycle arrest
by Shigella infection can be visualized in
the intestinal crypt progenitors of rabbit
ileal loops, and the IpaB-mediated arrest
contributes to the efficient colonization of
the host cells34. The finding that bacterial
activity can retard intestinal epithelial
renewal for the purpose of gaining an
infectious foothold adds a new facet to
bacterial infection.
Conclusions
In this Progress article, we have discussed
recent advances in our understanding of
the strategies that are used by Shigella to
infect the intestinal epithelium. During
basolateral entry into polarized epithelial
cells in the early stage of infection, Shigella
deliver a subset of effectors, including
IpaA, IpaB, IpaC, IpgB1, IpgB2, IpgD and
VirA, around the bacterial surface and
directly into the host cells through the T3SS
(TABLE 1). During multiplication within the
epithelium, Shigella secrete another subset
of effectors, including IcsB, VirA, OspF,
OspG and IpaH family proteins, again
through the T3SS, which enable the bacte-
ria to survive intracellularly, promote intra-
and intercellular movement and modulate
the host inflammatory response (TABLE 1).
Shigella then seem to directly access the
intestinal crypts and invade non-polarized
epithelial progenitor cells, in which they
deliver IpaB and slow the progression of the
cell cycle, thereby prolonging the lifespan of
their replicative niche.
From the work reviewed here and else-
where1–5, it is clear that bacterial effectors
have pivotal roles in the various aspects of
bacterial infection. Importantly, some of
the pathogenic functions of the effectors
that are involved in infection or in bacte-
rial defence against the host’s immune sys-
tem are occasionally shared by some other
pathogens (TABLE 1). Consequently, studies
of the roles of Shigella effectors (and effec-
tors from other bacteria) will contribute
to our understanding of host–bacteria
interactions as highly dynamic and widely
divergent biological events.
Michinaga Ogawa, Yutaka Handa, Hiroshi Ashida,
Masato Suzuki and Chihiro Sasakawa are at the
Department of Microbiology and Immunology, Institute
of Medical Science, University of Tokyo, 4‑6‑1,
Shirokanedai, Minato-ku, Tokyo 108‑8639, Japan.
16 | january 2008 | volume 6 www.nature.com/reviews/micro
Chihiro Sasakawa is also at the Department of Infectious
Disease Control, International Research Center for
Infectious Diseases, Institute of Medical Science,
University of Tokyo, 4‑6‑1, Shirokanedai, Minato-ku,
Tokyo 108‑8639, Japan and CREST, Japan Science and
Technology Agency (JST), Kawaguchi, 332‑0012, Japan.
Correspondence to C.S.
e‑mail: sasakawa@ims.u-tokyo.ac.jp
doi:10.1038/nrmicro1814  USA 102, 14046–14051 (2005).
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27. Arbibe, L. et al. An injected bacterial effector
targets chromatin access for transcription factor
NF‑κB to alter transcription of host genes involved
in immune responses. Nature Immunol. 8, 47–56
(2007).
28. Kramer, R. W. et al. Yeast functional genomic screens
lead to identification of a role for a bacterial effector
in innate immunity regulation. PLoS Pathog. 3,
179–190 (2007).
29. Zhang, J. et al. A Pseudomonas syringae effector
inactivates MAPKs to suppress PAMP-induced
immunity in plants. Cell Host Microbe 1, 175–185
(2007).
30. Nougayrede, J. P., Taieb, F., De Rycke, J. & Oswald, E.
Cyclomodulins: bacterial effectors that modulate the
eukaryotic cell cycle. Trends Microbiol. 13, 103–110
(2005).
31. Oswald, E., Nougayrede, J. P., Taieb, F. & Sugai, M.
Bacterial toxins that modulate host cell-cycle
progression. Curr. Opin. Microbiol. 8, 83–91
(2005).
32. Lara-Tejero, M. & Galan, J. E. A bacterial toxin that
controls cell cycle progression as a deoxyribonuclease
I‑like protein. Science 290, 354–357 (2000).
33. Gebert, B., Fischer, W., Weiss, E., Hoffmann, R. &
Haas, R. Helicobacter pylori vacuolating cytotoxin
inhibits T lymphocyte activation. Science 301,
1099–1102 (2003).
34. Iwai, H. et al. A bacterial effector targets Mad2L2, an
APC inhibitor, to modulate host cell cycling. Cell 130,
611–623 (2007).
35. Vodermaier, H. C. APC/C and SCF: controlling each
other and the cell cycle. Curr. Biol. 14, R787–R796
(2004).
36. Chen, Y., Smith, M. R., Thirumalai, K. & Zychlinsky, A.
A bacterial invasin induces macrophage apoptosis by
binding directly to ICE. EMBO J. 15, 3853–3860
(1996).
37. Nhieu, G. T., Caron, E., Hall, A. & Sansonetti, P. J.
IpaC induces actin polymerization and filopodia
formation during Shigella entry into epithelial cells.
EMBO J. 18, 3249–3262 (1999).
38. Zurawski, D. V., Mitsuhata, C., Mumy, K. L.,
McCormick, B. A. & Maurelli, A. T. OspF and OspC1
are Shigella flexneri type III secretion system
effectors that are required for postinvasion aspects
of virulence. Infect. Immun. 74, 5964–5976
(2006).
39. Miura, M. et al. OspE2 of Shigella sonnei is
required for the maintenance of cell architecture of
bacterium-infected cells. Infect. Immun. 74,
2587–2595 (2006).
40. Fernandez-Prada, C. M. et al. Shigella flexneri IpaH7.8
facilitates escape of virulent bacteria from the
endocytic vacuoles of mouse and human
macrophages. Infect. Immun. 68, 3608–3619 (2000).
Acknowledgments
This work was supported by a Grant-in-aid for the Scientific
Research on Priority Areas, Grant-in-aid-for Scientific Research
(C) and Grant-in-aid-for Young Scientists (B) from the Ministry
of Education, Culture, Sports, Science and Technology of
Japan (MEXT) and the Special Coordination Funds for
Promoting Science from Japan Science and Technology
Agency (JSTA).
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bradyrhizobium japonicum | Burkholderia pseudomallei |
Campylobacter jejuni | Coxiella burnetii | Edwardsiella ictaluri |
Escherichia coli | Helicobacter pylori | Legionella pneumophila |
Listeria monocytogenes | Mycobacterium marinum |
Mycobacterium tuberculosis | Pseudomonas syringae |
Rickettsia conorii | Salmonella typhi | Shigella dysenteriae |
Staphylococcus aureus | Streptococcus pyogenes
All links are active in the online pdf
 REVIEWS
The biology and future prospects of
antivirulence therapies
Lynette Cegelski*, Garland R. Marshall‡, Gary R. Eldridge§ and Scott J. Hultgren*
Abstract | The emergence and increasing prevalence of bacterial strains that are resistant to
available antibiotics demand the discovery of new therapeutic approaches. Targeting
bacterial virulence is an alternative approach to antimicrobial therapy that offers promising
opportunities to inhibit pathogenesis and its consequences without placing immediate life-
or-death pressure on the target bacterium. Certain virulence factors have been shown to be
potential targets for drug design and therapeutic intervention, whereas new insights are
crucial for exploiting others. Targeting virulence represents a new paradigm to empower the
clinician to prevent and treat infectious diseases.

Riboswitch
An mRNA control element
that changes conformation in
response to the binding of a
metabolite (for example,
glycine, lysine and
coenzyme B12) and influences
gene expression.
Microbiota
The entire collection of
microorganisms (bacteria,
archaea, fungi, sometimes
protozoa and viruses) that are
resident on or in the host.
*Department of Molecular
Microbiology, ‡Center for
Computational Biology and
Department of Biochemistry
and Molecular Biophysics,
Washington University, Saint
Louis, Missouri 63110, USA.
§
Sequoia Sciences, Saint
Louis, Missouri 63114, USA.
Correspondence to S.J.H.
e-mail: hultgren@borcim.
wustl.edu
doi:10.1038/nrmicro1818
Bacteria that are resistant to current antibiotics have
wreaked havoc in the clinic and are a primary cause of
death in the intensive-care units of our hospitals world-
wide1,2. Currently, most infections are caused by impor-
tant bacterial pathogens, such as Staphylococcus aureus,
that are penicillin resistant, and up to 50% are resistant
to stronger drugs, such as meticillin3. Meticillin-resistant
S. aureus (MRSA) infections have reached epidemic
levels this autumn (2007) in some parts of the United
States, and are spreading through many sports centres,
schools and gymnasiums, affecting predominantly stu-
dent athletes, but also younger schoolchildren, and have
already caused deaths in a matter of weeks. Moreover, as
most bacterial strains are becoming resistant to multiple
antibiotics, including vancomycin (the current drug of
choice for the treatment of MRSA), there is an urgent
need for antibiotics that have new modes of action on
therapeutic targets4,5.
Currently used antibiotics were derived by screening
natural products and compound libraries against whole
organisms, which identified compounds that have bac-
teriostatic or bacteriocidal activity. Analogues of these
parent drugs, which have improved potency, phar-
macological properties and efficacy against resistant
strains, have increased the number of antibiotics that
are clinically available6. However, the commercializa-
tion of new classes of antibiotics over the past 20 years
has not met expectations, and current pharmaceuti-
cal pipelines lack new, broad-spectrum antibiotics7–9.
The antibiotics that are available today are primarily
variations on a single theme — bacterial eradication
based on different modes of action at the molecular
level. Some target cell-wall biosynthesis, whereas others
inhibit protein synthesis or DNA replication. More
recently, fatty-acid biosynthesis has been proposed as
a viable bactericidal target. Lysine analogues have also
been identified that target lysine riboswitches and inhibit
bacterial growth10. Further study of such new targets is
an important element in the development of new drugs.
However, all these strategies target bacterial cellular
processes that are crucial for microbial survival. In our
current battle against infectious diseases, clinicians are
limited to the use of antibiotics that stimulate bacte-
rial evolution11,12. New tactics and weapons are needed
to combat bacteria that are, owing to evolution and
selection, moving targets.
Targeting bacterial virulence is an alternative
approach to the development of new antimicrobials
that can be used to disarm pathogens in the host13–15.
The overall strategy is to inhibit specific mechanisms
that promote infection and are essential to persistence
in a pathogenic cascade (for example, binding, inva-
sion, subversion of host defences and chemical signal-
ling), and/or cause disease symptoms (for example, the
secretion of toxins). Stripping microorganisms of their
virulence properties without threatening their existence
may offer a reduced selection pressure for drug-resistant
mutations. Virulence-specific therapeutics would also
avoid the undesirable dramatic alterations of the host
microbiota that are associated with current antibiotics.
Indeed, the microbial cells that comprise the microbiota
of a healthy human outnumber human cells by tenfold16;
they colonize distinct sites throughout the body and
confer numerous advantages to the host, some of which
are only beginning to be understood. The balance of
bacterial populations in the gut, for example, influences

nature reviews | microbiology volume 6 | january 2008 | 17

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REVIEWS

Table 1 | Representative adhesive fibres, fibre classification and disease association

Adhesive fibre Assembly proteins Adhesin Organisms Associated diseases
Fibres that use the chaperone–usher pathway
Type 1 pili FimC and FimD FimH Escherichia coli, Klebsiella pneumoniae and Cystitis
Salmonella species
P pili PapD and PapC PapG E. coli Cystitis and pyelonephritis
Prs pili PrsD and PrsC PrsG E. coli Cystitis
S pili SfaE and SfaF SfaS E. coli Urinary-tract infection and
newborn meningitis
Hif pili HifB and HifC HifE Haemophilus influenzae Otitis media and
meningitis
Type 2 and 3 pili FimB and FimC FimD Bordetella pertussis Whooping cough
Pef pili PefD and PefC Unknown Salmonella enterica serovar Typhimurium Gastroenteritis
(S. typhimurium)
Long polar LpfB and LpfC Unknown S. typhimurium Gastroenteritis
fimbriae
MR/K(type 3) pili MrkB and MrkC MrkD K. pneumoniae Pneumonia
Myf fimbriae MyfB and MyfC Unknown Yersinia enterocolitica Enterocolitis
Alternate chaperone pathway
CS1 pili CooB and CooC CooD E. coli Diarrhoea
Extracellular nucleation-precipitation pathway
Curli CsgB (nucleator), CsgE CsgA (major subunit) E. coli Sepsis
and CsgF (assembly) and
CsgG (secretion)
Tafi AgfB (nucleator) AgfA (major subunit) Salmonella enterica serovar Enteritidis Mouse typhoid
General secretion pathway
Type 4 pili General secretion PilC Neisseria gonorrhoea Gonorrhoea
apparatus
Pilin protein Pseudomonas aeruginosa, Vibrio cholerae, Cholera
Mycobacterium bovis and Dichelobacter nodosis
Gram-positive pili
Pili LPXTG-motif-mediated Unknown Corynebacterium diphtheriae, Streptococcus Pneumoccocal
export and covalent mutans and Streptococcus pneumoniae diseases
polymerization
Tafi, thin aggregative fimbriae.

Pilus
A non-flagellar filamentous
appendage that is formed on
the surface of many bacteria.
Quorum sensing
(QS). The process by which
bacteria use signalling
molecules to monitor bacterial
density and coordinate gene
expression in a population-
density-dependent manner.
Adhesin
The surface-exposed bacterial
molecule that mediates
specific binding to a receptor
or ligand on a target cell.
energy harvest and caloric intake through complex
interbacterial metabolic networks17. The microbiota
is dynamic, and shifts in the balance of microorgan-
isms that alter the sizes of different bacterial popula-
tions — for example, the use of traditional antibiotic
therapy — can lead to the loss of symbiotic benefits
and the proliferation of disease-causing opportunistic
pathogens.
A commitment to develop therapeutics that target
virulence requires a serious change in our perspective for
treating infectious diseases. Some elements of virulence
do seem to be fundamental for many pathogens, and
drugs that target these elements should exhibit broader-
spectrum activity. However, many antivirulence drugs
could be designed to target specific pathogens and the
virulence factors that are unique to their pathogenic
cascades. In addition, virulence-gene expression is a
function of time and space throughout infection, in
which factors such as pili could function early to medi-
ate adhesion, and others, such as toxin production and
quorum sensing (QS), could operate later, and in a different
niche, as bacteria replicate and respond to host defences.
As the coordinated regulation of virulence-gene expres-
sion is dynamic and location specific in the host, it
should be possible to identify and target vital genetic or
molecular bottlenecks — that is, to target the Achilles’
heel of a pathogen during infection18–20. Systems biol-
ogy is engaged in mapping the genetic control networks
and molecular correlates of pathogenesis (ideally, using
parameters obtained from relevant in vivo models) to
drive the discovery of these bottlenecks and identify
new drug targets.
Vaccination and other immunomodulatory strategies
are additional crucial avenues that are being pursued to
combat infectious disease and antibiotic resistance, both
in immunocompetent and immunologically compro-
mised hosts. The complex relationship between host
immunity and microbial pathogenesis, the balance
between protective immunity and immunopathology
and strategies to exploit the many networks that are
involved in antimicrobial strategies have been exten-
sively reviewed21–24 and will not be discussed in detail

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© 2008 Nature Publishing Group
 REVIEWS

Binding Pathogens are capable of presenting multiple adhesins

that can be expressed differentially to permit binding
in specific sites and at particular times over the course
of a complex infectious cycle. Thus, it may be difficult
Superficial to develop a universal class of anti-adherence drugs.
facet cell
Nevertheless, several specific pathogenic adhesive strat-
egies have emerged as hallmark requirements for viru-
Spread to new cells lence in certain infectious diseases that are immediate
Invasion and replication
targets for drug discovery and development.
Some bacteria present non-fimbrial adhesins
on their surface. These are expressed as monomeric
proteins or protein complexes that assemble at the
cell surface — for example, the Dr family of adhesins
Underlying that are expressed by Escherichia coli and are impor-
transitional cell
tant for adhesion in the intestine and urinary tract.
Adhesive autotransporters represent a class of afimbrial
adhesins that are expressed by various unrelated micro­
organisms, including species of Rickettsia, Bordetella,
Neisseria and Helicobacter and many members of the
Biofilm formation family Enterobacteriaceae. Haemophilus influenzae, a
causative agent of sinusitis, bronchitis and otitis media,
IBC expresses an adhesive autotransporter called Hap
that mediates binding to components of the host-cell
extracellular matrix.
Most adhesins, however, are incorporated into hetero­
polymeric extracellular fibres called pili or fimbriae.
Biomass dispersion Many distinct virulence fibres have been described
and cell exit in Gram-negative organisms25. Pili are also produced by
Figure 1 | Multi-step pathogenic cascade of uropathogenic Escherichia coli (UPEC). Gram-positive organisms (reviewed in REF. 26) and have
UPEC coordinate highly organized temporal and spatial events to Reviews
Nature colonize| Microbiology
the urinary been linked to virulence in Streptococcus pneumoniae27.
tract. UPEC bind to and invade the superficial facet cells that line the bladder lumen, Although bacterial fimbriae have diverse functions,
where they rapidly replicate to form a biofilm-like intracellular bacterial community many seem to be crucial to the binding and persistence
(IBC). In the IBC, bacteria find safe haven, are resistant to antibiotics and subvert of pathogenic microorganisms in the host (TABLE 1). In
clearance by host innate immune responses. UPEC can persist for months in a the following subsections, we review the importance of
quiescent bladder reservoir following acute infection and challenge current
pilus-mediated adhesion by E. coli in the pathogenesis
antimicrobial therapies. Quiescent bacteria can re-emerge from their protected
intracellular niche and be a source of recurrent urinary-tract infections. Insight into the
of urinary-tract infection (UTI) and discuss strategies
processes that accompany IBC formation and biofilm dispersal, as well as the factors to inhibit pilus biogenesis. We also describe the need for
that drive bacteria into the reservoir, may aid the design of preventive or therapeutic new approaches to prevent and treat UTI and examine the
strategies for recurrent infections. market considerations for antivirulence therapeutics.

Denying access to uropathogens. Uropathogenic E. coli

Autotransporter
A large family of secreted
proteins in Gram-negative
bacteria that harbour three
functional domains — the
amino-terminal signal peptide,
the secreted mature protein
(passenger domain) and a
carboxy-terminal translocator
domain — to allow secretion of
the passenger protein.
Biofilm
A community of cells that are
attached to a surface or
interface or to each other, and
are imbedded in a self-made,
protective matrix of
extracellular polymeric
substances.
here. In this Review, we highlight the diversity of the
bacterial virulence mechanisms and consider their
consequences in infectious disease. We review recent
efforts towards antivirulence-based drug discovery in
the framework of marketable drugs, and discuss the
challenges that remain and factors that are crucial to
developing the antivirulence therapeutic approach.
Microbial attachment and invasion
The physical interaction between the pathogen and the
host is crucial to the pathogenesis of virtually every
bacterial disease. Specific proteins called adhesins
mediate the adhesive interactions between the patho-
gen and a cell-surface ligand on the host cell that is
required for host colonization. The ability to impair
bacterial adhesion represents an ideal strategy to com-
bat bacterial pathogenesis because of its importance
early in the infectious process, and it is also suitable for
implementation as a prophylactic to prevent infection.
(UPEC) are the major causative agents of UTI and
engage in a coordinated and regulated genetic and mol­
ecular cascade to assemble type 1 and P pili, which are
associated with infections of the bladder and kidney,
respectively (reviewed in REF. 28). Virtually all clinical
UPEC isolates express type 1 pili29. These are required
to bind mannose-containing host receptors, invade
host bladder-epithelial (urothelial) cells 30,31 and
initiate a pathogenic cascade that involves several
distinct phases as examined in the mouse cystitis
model32,33 and human UTIs34 (FIG. 1). Within urothe-
lial cells, bacteria first replicate rapidly to form dense
biofilm-like communities and are protected from the
flow of urine and host defences33,35. UPEC eventually
detach and then disperse, or flux, from the intracel-
lular bacterial community (IBC) to initiate new rounds
of IBC formation in other cells. Some fluxing bacte-
ria form filaments, evade neutrophil phagocytosis
and facilitate bacterial survival33,35. Even after acute
infection is resolved, bacteria can remain within the

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© 2008 Nature Publishing Group
REVIEWS

Uroplakin E. coli

Type 1 pilus

Superficial facet cell

b
No compound Plus pilicide

N O CO2Li

Pilicide
Figure 2 | Targeting microbial adhesion. a | Pathogenic Escherichia coli use type 1 pili to bind to hexameric uroplakin
protein arrays on the surface of superficial facet cells that line the bladder lumen. Type 1 pili mediate the binding to, and
subsequent invasion of, these cells. b | Pyridone-based pilicides inhibit pilus biogenesis by disrupting chaperone–usher
Nature Reviews | Microbiology
protein interactions and dramatically reduce piliation levels. Image on the left in panel a reproduced, with permission,
from REF. 102  (1995) National Academy of Sciences. Image on the right in panel a reproduced, with permission, from
REF. 35  (1998) American Association for the Advancement of Science. Images in panel b reproduced, with permission,
from REF. 45  (2006) National Academy of Sciences.

Chaperone–usher system
A system that facilitates the
folding, transport and ordered
assembly of pilus subunits at
the cell surface.
bladder for many days to weeks, regardless of standard
antibiotic treatments, and can be implicated in recur-
rent infection36. It might be possible to target several
factors in the pathogenic cascade to inhibit virulence.
Type 1 pili represent an attractive drug target because the
bottleneck of invasion and IBC formation selects for fit-
ness in the urothelium and type 1 pili are required for
both events.
Two general strategies have emerged to inhibit
pilus-mediated function. The first is adhesion spe-
cific and involves physically precluding pathogen
binding to host cells. Carbohydrate derivatives of
host ligands, for example, have showed efficacy
in blocking the adhesive properties of both type 1
and P pili in biophysical and haemaglutination
assays 38–40. This approach can be readily extended
to other adherent organisms by tailoring the anti-
adhesive compounds to their receptor specificities
in vivo. The goal of the second strategy is to inter-
rupt pilus assembly, which also blocks pilus-mediated
adhesion as well as invasion and intracellular biofilm
formation.
Type 1 and P pili are both assembled by the chaperone–
usher system41. Although the fim and pap operons, which
are associated with type 1 and P pili, respectively, are
the best studied of these systems, 17 additional chaper-
one–usher operons have been identified in sequenced
E. coli genomes42. The chaperone–usher systems are
also necessary for the assembly of extracellular adhesive
organelles in a wide range of other pathogens, includ-
ing species of Salmonella, Pseudomonas, Haemophilus,
Klebsiella and Yersinia. Therefore, inhibitors of the
chaperone–usher system may serve as broader-range
therapeutics, an attractive feature that would enhance
the marketability of an effective drug. Pilicides are a
class of pilus inhibitors that target chaperone function
and inhibit pilus biogenesis43,44. A new class of pilicides,
based on a bi-cyclic 2‑pyridone scaffold, inhibit both
type 1 and P pili assembly in E. coli by targeting con-
served regions on chaperones that are ubiquitous in the
chaperone–usher pathways45 (fig. 2). These pilicides also
inhibit biofilm formation in E. coli. The synthetic pili-
cides disrupt an essential protein–protein interaction
between the chaperone and usher at a site that has been
identified by x‑ray crystallography. Thus, pilicides have
the opportunity to work either at the level of attachment
and invasion or the level of bacterial aggregation once
they are inside the superficial facet cells that line the
bladder lumen.
Therapeutic outlook for UTIs. The urinary tract is a com-
mon site of infection in humans, and UTIs result in more
than 8 million outpatient visits per year in the United States
and, in the year 2000, expended costs of US$3.5 billion
for evaluation and treatment 46,47. Women are the
most frequent sufferers — a female has a 50% chance

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© 2008 Nature Publishing Group
 REVIEWS

of developing an acute UTI during her lifetime — and a Toxin transcription

many experience recurrent infections. With advancing V. cholerae
age and co-morbidities, UTI becomes progressively
more common among both women and men, particu-
larly in the context of urinary catheterization. Limited
treatment options are available for patients with chronic Transcription
and recurrent UTIs. Although these patients can be initiator
treated using prolonged courses of antibiotics, this
Bacterial
radically disrupts the symbiotic host–microorganism toxin
balance and may be accompanied by the evolution of
drug-resistant organisms in the urinary tract48. In addi-
tion, UTIs have a strong causal correlation with systemic b Toxin trafficking and function
infection and sepsis if antibiotic therapy is ineffective49.
Because infection of the bladder is not limited to extra-
cellular colonization, and can involve both the invasion
of host cells and the subversion of host defences and
other specific mechanisms, such as filamentation, to
promote persistence and re-invasion, it may be essential
to target key bottlenecks, such as type 1 pili expression
and/or assembly. Current research efforts are underway
to identify other candidate nodes in the network of these
host–pathogen interactions.
The treatment of recurrent or chronic UTIs, in
which intracellular bacterial reservoirs already exist,
might require a synergistic therapy, such as combining Receptor
mimic
a pilicide, for example, with a stimulator of the immune
response or epithelial-cell renewal (to drive bacteria
out of intracellular niches). Scientific research and new Host
membrane
drug development for UTIs accompany a clear clinical
demand, and are associated with large patient popula-
tions for clinical-stage development and the potential
long-term profits that effective therapies will produce. Host toxin
We anticipate that drug-discovery and development receptor
efforts for UTIs and other chronic infections will
increase markedly over the next 5–10 years.

Bacterial toxins Intracellular

Toxin-powered pathogens can exert a devastating effect
from a distance. For many infectious diseases, the clinical
symptoms and cause of tissue damage can be attributed
to the action of secreted bacterial toxins. Botulinum,
anthrax, diphtheria, tetanus, cholera and Shiga toxins
are produced by distinct bacterial pathogens, and each
is a major cause of cellular malfunction and morbidity in
afflicted individuals50. In addition, their extreme toxicity
makes these toxins potential weapons for use in biologi-
cal warfare or a terrorist attack. Multiple opportunities
exist to prevent toxin damage to the host (FIG. 3).
toxin target
Intracellular
inhibitor
Figure 3 | Targeting toxin-powered pathogens. a | The
inhibition of toxin transcription, as described
Nature Reviews for Vibrio
| Microbiology
cholerae, is one way to inhibit the consequences of toxin-
mediated virulence. b | Neutralizing toxins, or preventing
their trafficking and/or enzymatic activity, at cellular
targets is an alternative strategy to inhibit toxin damage to
the host.

Targeting a toxin transcription factor. Vibrio cholerae
infection is characterized by severe diarrhoea and dehy-
dration, which becomes life threatening if effective treat-
ment of the symptoms is delayed. Cholera toxin provides
V. cholerae with its hallmark virulence and triggers the
debilitating symptoms of the disease by interacting with
G proteins and cyclic AMP in the intestinal lining to
interrupt proper ion transport, which results in mas-
sive fluid loss51. The potent toxin has been recognized
by Hung and colleagues52 as an ideal candidate for drug
development, which, as resistance has emerged to the
antibiotics of choice, ciprofloxacin and azithromycin,
is particularly needed. The small molecule virstatin
was discovered by screening for inhibitors of toxT gene
expression. ToxT is a transcription factor that activates
the gene transcription of both cholera toxin and another
V. cholerae virulence factor, the toxin-co-regulated pilus52.
Thus, virstatin inhibits cholerae-gene expression, the
earliest step in toxin production.
Selective inhibition of gene expression is a general
strategy that can be implemented for many virulence
factors, as expression is controlled by the environment
that is sensed by the organism. In the clinic, however,
such a therapy may have a short window of opportunity

nature reviews | microbiology volume 6 | january 2008 | 21

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REVIEWS
Botulism
A rare, but serious illness that
is caused by a nerve toxin,
botulinum, that is produced by
the bacterium Clostridium
botulinum.
Chemical genetics
The strategy of using small
molecules to alter and
interrogate biological
processes. The small-molecule
tools of dissection in this
approach harbour the precious
chemical scaffolds that may
lead directly to new
therapeutics.
Project BioShield
The Project BioShield Act was
incorporated into law by the
United States government in
July 2004. Through Project
BioShield, $5.6 billion will be
invested by 2013 in the
development of new
technological and therapeutic
countermeasures against
potential bioterrorism agents
and to purchase and stockpile
effective therapeutics to
prevent and treat the illnesses
that are related to these
threats.
22 | january 2008 | volume 6 www.nature.com/reviews/micro
to work. On the one hand, after an extended time, bac-
teria may reach high numbers and produce sufficient
toxin to overwhelm the host, such that inhibition of toxin
production alone may be too late. On the other hand,
effective and selective therapeutics could be useful
prophylactically in limiting epidemics.
Neutralizing toxins using antibodies. An antivirulence
strategy has been the treatment of choice for the post-
exposure treatment of tetanus, diphtheria and botulinum
toxins. Antibodies against the toxins are administered to
patients to attempt to neutralize toxins while infection
is cleared53. In addition to simply neutralizing toxins,
benefit to the host may be ascribed, in part, to a more
sophisticated host response that includes the antibody-
mediated recognition and potential clearance of patho-
gens and their toxic cargo, as reviewed elsewhere22–24. The
neutralizing antibodies are typically obtained from the
sera of immunized horses or humans, but the produc-
tion of monoclonal antibodies is an attractive alternative.
Many monoclonal-antibody therapies are already in use
for preventing transplant rejection and oncological treat-
ment; an antiviral monoclonal antibody, palivizumab,
which prevents respiratory syncytial virus in children,
was licensed in 1998.
Currently, adult cases of botulism, a rare infectious
disease that is caused by Clostridium botulinum, are
treated with an antitoxin that contains horse antibod-
ies raised against type A, type B and/or type E strains
of botulinum neurotoxins. This drug is available from
the Centers for Disease Control and Prevention (CDC),
United States, if specially requested by a physician. It is
listed in the Official Monographs of the United States
Pharmacopeia, even though it has never been examined
in controlled human clinical trials. Owing to the equine
antitoxin’s potentially serious side effects, it has not typi-
cally been used to treat infants suffering from botulism.
However, a landmark in antivirulence therapy occurred
on 23 October 2003. The Food and Drug Administration
in the United States licensed an antivirulence drug that
contained antibotulinum-toxin antibodies produced
from humans to the California Department of Health
Services, based on their seminal clinical research that
spanned approximately 15 years. A placebo-controlled
clinical trial consisting of 122 infants with botulism
demonstrated a profound clinical benefit for patients
treated with human botulism immune globulin (BIG),
subsequently named BabyBIG54. Clinical results showed
a reduction in the length of hospital stay, duration of
intensive care, duration of mechanical ventilation, dura-
tion of tube or intravenous feeding and hospital charges
per patient. This remarkable clinical evidence strongly
supports a treatment strategy of neutralizing bacterial
toxins post-infection.
Targeting toxin trafficking and function. Antibody-
neutralization strategies will fall short for some patients
and infectious diseases. Toxins that are delivered
through the type III secretion system (T3SS; discussed
below), for example, are delivered directly into host cells,
do not circulate globally and are not ideal candidates
© 2008 Nature Publishing Group
for antibody neutralization. The best defences against
toxin-empowered pathogens that are encountered in the
community, or as potential bioweapons, might require
a better understanding of pathogenesis and toxin bio-
chemistry. For many toxins, new research is needed to
provide higher-resolution models of toxin trafficking
than that presented in FIG. 3 (Ref. 55). In this regard,
chemical genetic approaches are driving the discovery
of toxin-trafficking inhibitors and, in turn, these small
molecules are being used as tools, or molecular scalpels,
to dissect the mechanistic details of toxin trafficking.
Selective luciferase-based screening assays, for example,
have identified lead compounds that inhibit the actions
of E. coli’s Shiga toxin once it has gained access to the host
cell, and therefore permit protein synthesis56,57. These
compounds stop the toxin in real time along its path
from the cell surface to the endoplasmic reticulum, and
provide a unique opportunity to examine toxin-transport
processes in more detail. The ability to target toxins after
they have entered host cells, but before they exercise their
function, provides an additional opportunity in time and
space to control the sequelae of toxin action.
Improved models of the assembly and function of
anthrax toxin, produced by the Gram-positive Bacillus
anthracis, provide several distinct checkpoints that can be
targeted for interception55. Peptides and small-molecule
inhibitors have been identified that bind to lethal factor
(one of the three anthrax toxin components) and inhibit
the in vitro enzymatic activity that is linked to patho-
genesis in vivo58–60. Several molecules have been reported
to block endosomal acidification, and so inhibit the
conformational changes that are required for pore for-
mation at the host-cell surface, and anthrax-toxin entry.
Having multiple ways to target toxin-powered pathogens
encourages the development of new and diverse thera-
peutics that promise to treat a large number of infectious
diseases.
Focus on bioterrorism. The dangers that are associated
with an anthrax bioterrorism threat were made clear in
2001 when B. anthracis was disseminated throughout the
United States postal system leaving five dead and many
ill. Anthrax toxins, along with botulinum toxins, are
classified in the highest risk category of biological war-
fare agents by the CDC, and nearly $6 billion have been
allocated by Project BioShield to develop and stockpile
antibiotics, antitoxins and vaccines for these and other
high-threat agents61. Vaccination efforts are not generally
promoted, however, because the chance of exposure to
any particular bioterrorism agent is remote.
PharmAthene was founded in 2001 to develop
technologies and products to address these biosecu-
rity needs. Their new antivirulence drug, Valortim,
is a human monoclonal antibody that has demon-
strated prophylactic and, separately, post-infection
protection against B. anthracis toxins in rabbit and
non-human primate models 62. It has successfully
completed Phase I safety and pharmacokinetic evalu-
ation. In 2006, Cangene Pharmaceuticals received a
5-year, $362-million contract from Project BioShield
to develop and produce 200,000 doses of a potentially

Haemolytic uraemic
syndrome
A disease that primarily affects
infants and children and is
characterized by the loss and
destruction of red blood cells.
Occurs most commonly in
children after a gastrointestinal
infection or upper respiratory-
tract infection and can lead to
kidney failure.
nature reviews | microbiology volume 6 | january 2008 | 23
improved botulinum antitoxin, a heptavalent antitoxin
that is derived from horses (containing antibodies to
7 toxin subtypes), because of its potential use as a
bioterror agent. Its initial order was formally received
into the United States Strategic National Stockpile in
September 2007.
Scientific leaps and commercialization hurdles.
Antibody-based toxin neutralization (discussed above),
could be considered to be a simple approach to target
toxin-based virulence that does not require a high-
resolution roadmap detailing toxin trafficking and sub-
cellular targets. However, toxin-associated diseases are
often not that simple. They can be extremely complex,
with cascading symptoms that may or may not lead to
life-threatening complications. Consequently, the Anti-
Infective Drugs Advisory Committee meeting held on
12 April 2007, when considering the operational details of
executing clinical trials to demonstrate the effectiveness
of a new antivirulence therapy, emphasized the pitfalls
and hurdles that might impede the commercialization
of any antivirulence therapy that targets small patient
populations63. At this meeting, research results were
presented for two separate monoclonal antibodies that
are under development to treat Shiga-toxin-producing
bacterial infections. Shiga toxin is produced by Shigella
dysenteriae and Shiga-toxin-producing E. coli (STEC),
which includes the prototype strain E. coli O157:H7 that
has caused several food-borne outbreaks around the
world. The toxin is secreted into the host cell and inhibits
host protein synthesis, which can lead to multiple clinical
manifestations, such as gastrointestinal disease, bloody
diarrhoea, the destruction of red blood cells and plate-
lets, and haemolytic uraemic syndrome64. Treatment using
antibiotics is controversial owing to the bolus of toxin
that could be released as bacteria die, which, potentially,
could overwhelm the patient’s defences.
Even though evidence of efficacy from animal models
was presented at this meeting, representatives from com-
panies and the physicians on the advisory committee
were unable to successfully construct an appropriate
clinical-trial design to evaluate these potential drugs.
The two main challenges for the successful completion
of clinical trials seem to be, first, that primary end-
points for this specific disease in humans are unclear
and, second, that achieving statistical significance may
not be attainable because the low incidence of this dis-
ease prevents adequate patient enrolment. The findings
of this meeting strongly suggest that researchers should
target the virulence mechanisms that are involved in
infectious diseases with clear and measurable clinical
end-points in sufficiently large patient populations.
These realities of commercialization seem to make
developing drugs against STEC and other complicated
diseases that affect small patient populations less attrac-
tive compared with diseases that affect large patient
populations, such as UTIs and chronic ear and sinus
infections. These hurdles will accompany the develop-
ment of future antivirulence products, and imminent
decisions surrounding the potential Shiga drugs will
influence the course of future endeavours.
© 2008 Nature Publishing Group
REVIEWS
Type III secretion
The Shiga and cholera toxins discussed above are
exported by type II secretion, a general secretion system
through which extracellular enzymes are also secreted65.
However, other pathogens deliver their virulent cargo
using the T3SS. This system orchestrates the export and
delivery of virulence factors, or effector proteins, from
the bacterial cytoplasm across the inner membrane, the
peptidoglycan and outer membrane and, finally, through
the host-cell plasma membrane, directly into the host-
cell cytosol in the manner of a molecular syringe66.
The T3SS machinery is used by many Gram-negative
pathogens, such as E. coli, Salmonella enterica serovar
Typhimurium, Shigella flexneri, Pseudomonas aeruginosa
and species of Yersinia and Chlamydia. Yersinia species,
such as Yersinia pestis, the causative agent of plague,
inject effector proteins called YOPs (Yersinia outer pro-
teins) through the T3SS into host cells to inhibit the host
immune response and help them to evade a potent
host response and subsequent clearance67,68.
The conserved elements in the T3SS machinery that
allow the interchange of T3SS components among some
bacteria69,70 suggest that it may be possible to design
broad-range T3SS inhibitors that are effective in treat-
ing disparate pathogens, regardless of the unique effec-
tor proteins that they deliver. Small-molecule inhibitors
of the T3SS have been described in Yersinia pseudotu-
berculosis71,72. More recently, the discovery that small-
molecule T3SS inhibitors in Yersinia spp. inhibit the
T3SS in Chlamydia trachomatis supports the notion of
developing broader-range T3SS inhibitors. Interestingly,
the role of the T3SS in the pathogenesis of Chlamydia
spp. infection is not well understood, owing, in part, to
the inability to manipulate Chlamydia spp. genetically.
However, in the previously mentioned study, the small-
molecule inhibitors were recruited using chemical
genetics to assess the potential importance of the T3SS in
C. trachomatis infection. Treatment with the T3SS
inhibitor did inhibit the in vivo pathogenic cascade
and resulted in a decrease in secreted effector proteins,
suggesting that the T3SS was inhibited and is essential
to the C. trachomatis infectious cycle73–75. These T3SS
inhibitors will be a useful tool for further study of the
T3SS in Chlamydia spp., and might represent target
leads for future therapeutics.
Biofilms and chronic infections
Biofilms are complex, organized bacterial assemblies
that are highly resistant to antibiotics and host defences.
Biofilms can form on abiotic surfaces, such as surgical
implants and catheters, and result in persistent infec-
tions that are difficult to treat, thereby leading to further
health complications and longer hospital stays. Bacteria
in chronic wounds, which are particularly prevalent in
the elderly and diabetic populations, form biofilms that
prevent proper healing76. Indeed, the ability of bacteria
to persist robustly in biofilm communities in the human
host and the environment, even against antibiotic pres-
sure, is a fundamental observation that extends across the
field of microbiology and poses serious challenges to
the control and treatment of chronic infectious disease.
REVIEWS

Table 2 | Selected two-component response systems (TCRSs) involved in virulence

Histidine-kinase sensor and Organisms
response regulator
AgrC; AgrA Staphylococcus aureus and
Staphylococcus epidermidis
AlgD; AlgR Pseudomonas aeruginosa
RcsC; RcsB Escherichia coli
BvgS; BvgA Bordetella pertussis
PhoQ; PhoP P. aeruginosa, Salmonella
enterica serovar Typhimurium, lipopolysaccharide and resistance to antimicrobial
Yersinia pestis and Vibrio
cholerae
TCRSs involved in the regulation of resistance to antibiotics
VanS; VanR Enterococcus faecalis
VncS; VncR Streptococcus pneumoniae
RprX; RprY Bacteroides fragilis
Associated virulence property
Control of most aggressive virulence factors
Alginate
Capsule
Toxins, adhesins and colonization factors
Divalent cation sensing, modification of
peptides
Vancomycin resistance
Vancomycin resistance
Tetracycline resistance

The intracellular biofilms that are formed by UPEC Quorum sensing

in the urinary tract after their invasion of the bladder
epithelium and smaller numbers of quiescent bac-
teria that are harboured in underlying reservoirs
are implicated in the aetiology of recurrent UTIs36.
Clinical studies of recurrent infection of the middle-
ear tissue in children indicate that chronic otitis
media stems from a persistent biofilm following the
first infection77. Helicobacter pylori biofilms have been
documented in the biopsied gastric mucosa of patients
suffering from gastric ulcers, and it has been suggested
that the ulcers are manifestations of these biofilms 78.
In addition, patients with cystic fibrosis are threatened
by P. aeruginosa biofilm-mediated chronic lung infec-
tions, which are responsible for the high morbidity and
mortality of patients with cystic fibrosis79,80.
Several strategies have emerged to inhibit biofilm
formation and eradicate established biofilms. These
include, but are not limited to: preventing the initial
adherence of bacteria to either a surface or another
bacterium (discussed above); interrupting QS mech-
anisms that are required for the gene expression of
biofilm components (discussed below); inhibiting the
biosynthesis of the integral polysaccharide and pro-
teinaceous extracellular components of the biofilm
matrix81; and identifying or tailoring enzymes that
can degrade the biofilm matrix, thereby rendering
bacteria accessible to traditional antibiotics and/or
immune clearance.
If we examine the anti-infective marketplace, the
health and economic impact of multidrug-resistant
organisms, particularly in the hospital setting, is
driving the primary market for drug development.
However, markets that encompass recurrent and
chronic bacterial infections consist of significantly
larger patient populations that have considerable
unmet clinical needs and provide attractive opportu-
nities for drug-development efforts. Effective biofilm
inhibitors could dramatically change treatment regi-
mens for many infectious diseases and benefit large
patient populations.
QS is the illustrative term that is used to describe the
chemical signalling that takes place among bacteria to
keep track of their cellular density. QS is mediated by the
production and subsequent recognition of small molecules
called autoinducers, and is used to coordinate gene expres-
sion and regulate the numerous processes that are involved
in community behaviour and virulence, for example,
motility and biofilm formation (reviewed in Refs 82,83).
In nature, the red seaweed Delisea pulchra produces
chemical compounds called furanones that intercept QS
signals and prevent microbial colonization84,85. This natural
phenomenon has inspired the search for compounds that
selectively inhibit QS, biofilm formation and the virulence
of human pathogens during infection86.
P. aeruginosa is frequently encountered in noso-
comial infections and lung infections in patients with
cystic fibrosis, and produces more than 30 QS‑regulated
virulence factors. Numerous studies, predominantly car-
ried out over the past 5 years, strongly support the notion
that QS inhibitors, including isolated and synthetic
furanones, can be effective in treating bacterial infec-
tions in vivo87,88. Notably, P. aeruginosa biofilms exhibit
increased susceptibility to sodium dodecyl sulphate
and the antibiotic tobramycin if treated with a synthetic
furanone, compound C‑30 (Ref. 87). In a mouse pulmo-
nary infection model, treatment with compound C‑30
resulted in increased bacterial clearance87. Thus, inhibi-
tion of QS can increase the susceptibility of biofilm bac-
teria to host defences and antibacterial agents. Givskov
and colleagues87 have suggested that direct targeting of
QS might be effective as an early prophylactic treatment
of individuals who have P. aeruginosa infections in the
lungs, on implants or in wounds.
Other examples underscore the challenge of interrupt-
ing the right signals at the right times to reduce bacterial
virulence in the host. A non-native N‑acylated‑l-homo-
serine lactone, for example, was recently discovered that
can either inhibit or strongly induce QS in the marine
symbiont Vibrio fischeri, depending on the molecule
concentration89. Results from a panel of compounds in the

24 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group

AIP
AgrC 1 sequenced bacterial genomes92,93. Selected TCRSs that
AgrB
Cytoplasm
Histidine- 2 generally consist of receiver domains that are covalently
kinase P linked to effector domains (FIG. 4). Specifically, each
sensor 3
AgrA Response regulator
Nucleus
4 as the interfaces of homodimer formation of activated
P
AgrA
Gene RNAIII agrB agrD agrC agrA
regulation
Figure 4 | Pairing quorum sensing and two-component signalling in the
Nature Reviews | Microbiology
staphylococcal agr system. Staphylococcus aureus uses a two-component response
system (TCRS) to mediate quorum sensing (QS). The regulation of QS involves the
production of an autoinducer and an increase in its concentration, expression of RNAIII
and the subsequent regulation of QS genes. S. aureus produces an autoinducing peptide
(AIP) that accumulates extracellularly and activates the TCRS. The TCRS involves signal
recognition by a histidine kinase (AgrC) (1), followed by histidine phosphorylation (2) and
phosphotransfer to a response regulator (AgrA) (3), which then binds to the RNAIII
transcript that encodes a small RNA that functions to modulate gene expression of
S. aureus genes (4).
same family elicited varied QS responses, which implies
that the rational design of modified QS antagonists or
agonists may not be straightforward; new insights are
needed to fully understand these responses. In addition,
redundancies among QS systems can render inhibitors
of a single system ineffective. Indeed, two of the three
parallel QS systems in V. cholerae are dispensable for host
colonization and the production of two hallmark viru-
lence factors, cholera toxin and the toxin co-regulated
pilus90. QS circuits are being examined in numerous
models to determine if similar redundancies exist and to
what extent individual chemical signals influence multi-
ple QS pathways. The generation of QS circuit diagrams
may reveal viable drug targets and small molecules
that may prove useful as scalpels to probe the roles of QS
in different systems.
Two-component response systems
A central requirement of bacterial virulence is the ability
to express subsets of genes in response to signals that are
specific for a particular environment. Two-component
response systems (TCRSs) seem to be the dominant
mechanism by which bacteria and fungi respond to
nature reviews | microbiology volume 6 | january 2008 | 25
© 2008 Nature Publishing Group
REVIEWS
their environment91. TCRSs can control host invasion,
drug resistance, motility, phosphate uptake, osmoregula-
tion, nitrogen fixation and other functions. More than
4,000 TCRSs have been identified in approximately 400
regulate virulence are provided in TABLE 2.
The essence of signal transduction lies in the recogni-
tion and interpretation of environmental signals that are
related to host infection, and conversion of those signals
into specific protein–protein interactions and tran-
scriptional activation94. Bacterial transduction systems
TCRS is composed of a histidine kinase that is activated
by extracellular signals in the host environment and a
response regulator that, in turn, transmits the signal to
the intracellular target to modulate the gene expression
of virulence factors. The interfaces between response
regulators and their protein modulators can be targeted
to prevent phosphorylation and/or activation, as well
response regulators that are required for the control of
gene expression. Broad-spectrum inhibitors could non-
selectively target the TCRSs that are common to many
bacteria. More selective inhibitors could target the inter-
action of specific phosphorylated response regulators to
prevent expression of a specific virulence gene95.
A TCRS is an integral component of the QS system in
Gram-positive bacteria that responds to bacterial density,
and is named agr for accessory gene regulator. The global
regulator agr controls the expression of most virulence
genes in staphylococci and is activated by secreted
auto­inducing peptides (AIPs) called thiolactones that
comprise 7–10 amino acids96. AgrC and AgrA are the
histidine-kinase sensor and response regulator, respec-
tively, of the TCRS. AIP thiolactones have been suggested
as leads for the development of S. aureus therapeutics that
are urgently needed, owing to the fact that nosocomial
and community-acquired MRSA infections are on the
rise. The agr system also functions to downregulate fac-
tors that are important in biofilm formation, and agr
dysfunction is associated with increased biofilm produc-
tion97. As discussed above, a more detailed understanding
of the organism’s control networks is needed to identify
the ideal genetic or molecular checkpoints that need to
be targeted to reduce virulence in the host.
The prevention of virulence mechanisms that promote
antibiotic tolerance could also improve the efficacy of cur-
rent antibiotics, and, particularly, slow down the processes
that lead to drug resistance. Several TCRSs are responsible
for inducing the gene expression that confers bacterial tol-
erance to antibiotics. Vancomycin resistance, for example,
is triggered by the VanR–VanS TCRS. VanS detects the
glycopeptide antibiotic and VanR activates the expression
of the enzymes VanA, VanH and VanX, all of which are
required for resistance98,99 (reviewed in REF. 11). The TCRS
proteins and the three enzymes cooperate to synthesize an
altered peptidoglycan framework that is tolerant to vanco-
mycin exposure, which is a hallmark of the vancomycin-
resistant enterococci and vancomycin-resistant S. aureus
that have emerged in the clinic.
REVIEWS

Conclusions and perspectives In battling infection, disabling microbial virulence

This era may come to be remembered as one in which
infectious diseases made a dramatic worldwide resurgence,
owing to the rise of antibiotic resistance and emergence of
new diseases. We must increase the number of available
therapeutics, protect the effectiveness of current antibiot-
ics and, importantly, decrease further pressure for the evo-
lution of new drug-resistance mechanisms. Neutralizing
bacterial toxins by using antibodies has emerged as the
most-pursued antivirulence therapeutic strategy in indus-
try, with at least six candidates undergoing clinical trials.
The initial successes of these antitoxins seem to provide
empirical evidence that supports increased research into
other antivirulence approaches. Therefore, it is impera-
tive that we determine the mechanisms of virulence and
the consequences of host–pathogen interactions from
both the pathogen and host perspective. Insights regard-
ing bacterial virulence and pathogenesis have emerged,
yet many crucial questions remain unanswered. Major
breakthroughs will require multi-disciplinary tools
from bacterial and host genetics, structural biology and
in vivo imaging, animal models, cell biology, immunol-
ogy, biochemistry, chemical genetics, functional genom-
ics and systems biology. Atomic-level details of the
structural interactions at host–pathogen interfaces are
vital for the discovery of effective antivirulence drugs by
structure-based drug design for proposed targets.
in vivo could shift the advantage to the host and render
bacteria impaired in defeating host defences. Alternatively,
antivirulence drugs could be used in combination therapy,
in which bacterial clearance is mediated by standard anti-
biotics and the symptoms of virulence are suppressed. The
effective deployment of antivirulence drugs will require
rapid diagnosis in the clinic of the organism (or organ-
isms) that is responsible for infection and may include
profiling of its virulence gene or genes100. Such routine
and rapid diagnosis would also improve the use of stand-
ard antibiotics, and represents a necessary investment as
medicine improves and becomes more personalized.
We must succeed in this continuous war against
infectious disease. A recent review of the antibacterial
drug-development efforts by GlaxoSmithKline reveals, in
essence, that we have underestimated our opponent and
that arrogance has dominated the search for new antibi-
otics101. Regaining our competitive advantage will require
us to see beyond what we currently accept as dogma. It
will also depend on the removal of perceived obstacles to
infuse the industry with new opportunities. We need to
accept that a one-drug-fits-all strategy will probably fail.
We also need to consider each specific disease, pathogen
and virulence mechanism, and combine the strengths
of synergistic therapies to minimize the evolution by
pathogens of resistance.

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Acknowledgements
The authors acknowledge funding from the National Institutes
of Health to S.J.H. (grant numbers P50-ORWH/DK64540,
R01AI029549, R01AI048689 and R01DK51406), G.R.M.
(grant number R01GM068460) and L.C. (grant number
T32A107172).
Competing interest statement
The authors declare competing financial interests: see web
version for details.
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bacillus anthracis | Chlamydia trachomatis | Clostridium
botulinum | Escherichia coli | Haemophilus influenzae |
Helicobacter pylori | Pseudomonas aeruginosa | Shigella
dysenteriae | Shigella flexneri | Staphylococcus aureus |
Streptococcus pneumoniae | Vibrio cholerae | Vibrio fischeri |
Yersinia pestis | Yersinia pseudotuberculosis
FURTHER INFORMATION
Scott J. Hultgren’s homepage: http://hultgren.wustl.edu/
public
All links are active in the online pdf
REVIEWS

Getting organized — how bacterial

cells move proteins and DNA
Martin Thanbichler* and Lucy Shapiro‡
Abstract | In recent years, the subcellular organization of prokaryotic cells has become a focal
point of interest in microbiology. Bacteria have evolved several different mechanisms to target
protein complexes, membrane vesicles and DNA to specific positions within the cell. This
versatility allows bacteria to establish the complex temporal and spatial regulatory networks
that couple morphological and physiological differentiation with cell-cycle progression. In
addition to stationary localization factors, dynamic cytoskeletal structures also have a
fundamental role in many of these processes. In this Review, we summarize the current
knowledge on localization mechanisms in bacteria, with an emphasis on the role of polymeric
protein assemblies in the directed movement and positioning of macromolecular complexes.

*Max Planck Institute for
Terrestrial Microbiology,
Karl-von-Frisch-Straße,
35043 Marburg, Germany.
‡
Department of
Developmental Biology,
Stanford University School of
Medicine, Beckman Center
B300, 279 Campus Drive,
Stanford, California 94305,
USA.
Correspondence to M.T.
e-mail: thanbichler@
mpi-marburg.mpg.de
doi:10.1038/nrmicro1795
Published online
3 December 2007
Bacteria are among the most successful and widespread
organisms on the Earth. In the course of several billion
years of evolution, they have adapted to almost every
possible biological niche and developed an astonishing
range of metabolic pathways, life cycles and cell mor-
phologies. As a result of continuous selection for fast
reproduction rates, they have adopted highly stream-
lined architectures and small genomes that have high
coding densities. Their straightforward organization
has frequently been regarded as indicative of a primitive
cellular state. Consequently, advanced features, such as
cytoskeletal structures, intracellular transport proc-
esses and spatially regulated transcription and protein
localization, were traditionally thought to be restricted to
eukaryotic cells. However, this simplistic view was difficult
to reconcile with the complexity of many processes that
are found in bacteria. Notably, regulation of cell shape, cell
polarization and asymmetric cell division are common
phenomena in bacterial development that are unlikely to
occur without dedicated temporal and spatial regulatory
systems or dynamic cytoskeletal elements.
Recent work, driven by technological advances
that have facilitated the resolution of structural details
within bacterial cells, has revealed that bacteria have
indeed evolved mechanisms to actively control cell-cycle
progression and morphological differentiation. Some
of the factors that are involved in these processes are
homologues of well characterized eukaryotic proteins,
but they are frequently used outside of their established
functional contexts. Other factors are found exclusively
in bacteria and constitute novel regulatory and struc-
tural systems that have evolved to meet the specific
physiological requirements of the bacterial cell. In this
Review, we highlight key mechanisms of cellular organi-
zation in bacteria that reflect the striking momentum
that bacterial cell biology has gained in recent years.
Specifically, we will discuss pathways that mediate the
assembly and positioning of localized protein com-
plexes and set the foundation for temporal and spatial
regulatory processes within bacterial cells. In this
context, emphasis will be placed on the growing number
of dynamic cytoskeletal elements that have been iden-
tified in bacteria and their pivotal roles in subcellular
organization, DNA segregation and cell division.
Subcellular organization in bacteria
The realization that bacteria use specifically localized
protein complexes to orchestrate cellular processes led to
a surge of research on the mechanisms that structure the
bacterial cell (BOX 1). Among the many proteins with une-
ven subcellular distribution that have been identified in
recent years, two major classes can be distinguished. One
group forms largely stationary complexes that localize to
precisely defined subcellular positions, even though their
subunits might be in rapid exchange. Proteins belonging
to the second class, by contrast, are part of highly dynamic
interaction networks with components that continuously
change their subcellular location in a temporally and
spatially controlled manner.
Assembly of stationary protein complexes. Most sta-
tionary complexes that have been characterized so far
are based on integral membrane proteins. They usu-
ally assemble at the new pole (resulting from the most

28 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group

Forespore recent division), the old pole, the cell-division site or the
Precursor of the spore; a forespore septal membrane in the case of Bacillus subtilis
resting cell that is highly sporulation, where they mediate the establishment of
resistant to a number of cellular organelles, perform localized catalytic activities
environmental stresses, such as
heat, ultraviolet irradiation and
or serve regulatory functions. But how do these proteins
desiccation. assume a defined intracellular position? Evidence has
been presented that at least two localization mechanisms
Single-molecule tracking operate in bacteria: diffusion and capture; and targeted
Microscopic analysis of the
membrane insertion. During diffusion and capture,
movement of individual
fluorescently labelled
newly synthesized proteins are first inserted randomly
molecules within a cell. throughout the cytoplasmic membrane and then, after
diffusion, they are captured by an interaction with a pre-
viously localized membrane complex1,2. Targeted mem-
brane insertion, by contrast, describes a process whereby
a protein is delivered directly to its destination by trans-
location to a given cellular site. In all cases, a crucial issue
is the identity of the determinant that is responsible for
the localization of the membrane protein and how this
determinant is positioned in the first place.
Box 1 | Model systems in bacterial cell biology
Although an increasing number of species are studied with respect to their cell biology,
most information on cellular organization in bacteria is currently based on studies in
Escherichia coli, Bacillus subtilis and Caulobacter crescentus. The characteristics of these
model systems are listed below.
E. coli
• Long history as a bacterial model organism.
• Large collection of genetic tools, mutant strains and methods.
• Extensive body of knowledge on many aspects of its physiology.
B. subtilis
• Sporulation as a model for cellular differentiation.
• Well-studied physiology.
• Large size, which facilitates the resolution of cytoskeletal structures.
C. crescentus
• Asymmetric cell division (see the figure).
• One round of chromosome replication per cell division.
• Abundance of dynamically localized regulatory protein complexes.
• Synchronizability.
In the course of its life cycle, C. crescentus differentiates from a mobile, flagellated
swarmer cell into a sessile stalked cell (see the figure). Subsequently, it undergoes an
asymmetric cell division that regenerates the stalked cell and creates a new swarmer
cell. The stalked cell immediately enters a new round of cell division, whereas the
swarmer cell needs to develop into another stalked cell to start the next division cycle.
The single polar flagellum is shed during the swarmer-to-stalk-cell transition and
re-established at the pole opposite the stalk in the predivisional cell.
Swarmer cell
Stalked cell
Stalk
Flagellum
Nature Reviews | Microbiology
nature reviews | microbiology volume 6 | january 2008 | 29
© 2008 Nature Publishing Group
REVIEWS
Diffusion and capture. During B. subtilis sporula-
tion many proteins localize differentially to the septal
membrane, which provides an amenable system for
investigating the principles that underlie directed pro-
tein positioning within the cell (FIG. 1a). SpoIVFB, a pro-
protein-processing enzyme, is synthesized in the mother
cell, but must be localized in the septal membrane to
carry out its function. Rudner and colleagues1 reasoned
that SpoIVFB is either targeted to the septal membrane
directly upon synthesis or is first inserted randomly
around the mother-cell cytoplasmic membrane and
then, upon diffusion, captured at the septal mem-
brane. To distinguish between these two possibilities,
SpoIVFB was expressed from an inducible promoter
in vegetatively growing cells, in which it was found to
be randomly positioned in the cytoplasmic membrane.
During initiation of sporulation in these cells, and in the
absence of the inducer, the existing SpoIVFB accumu-
lated at the septum, demonstrating that diffusion and
capture is the probable mode of SpoIVFB localization
(FIG. 1b). Additional evidence that SpoIVFB diffuses to
its site of action was provided by taking advantage of
the fact that, when engulfment occurs, the membrane
surrounding the forespore is separated topologically
from the mother-cell cytoplasmic membrane from
which it is derived1. SpoIVFB that was synthesized after
engulfment was specifically enriched in the mother-cell
cytoplasmic membrane, but was absent from the outer
forespore membrane, again suggesting that diffusion
and capture, rather than targeted insertion, is the mode
of SpoIVFB localization.
Evidence for the diffusion-and-capture mode of pro-
tein localization also comes from single-molecule tracking
of the Caulobacter crescentus membrane histidine kinase
PleC, which localizes dynamically to the pole that is
opposite the stalk at specific times in the cell cycle2.
Following the movement of a PleC–green fluorescent
protein (GFP) fusion protein over a timescale of seconds
revealed two diffusion coefficients: a high coefficient for
molecules that were without directional bias and a lower
coefficient for molecules that were targeted to the cell
pole. However, the components that mediate the sink at
the cell pole have not yet been identified.
Diffusion and capture might be a widespread mecha-
nism that operates in several different systems. For
example, many components of the bacterial cell-division
apparatus are known to assume an even subcellular dis-
tribution unless a defined precursor complex has assem-
bled to capture them at mid-cell3. The same principle may
also apply to soluble proteins, which can be tethered to
a pre-localized targeting factor after three-dimensional
diffusion through the cytoplasm or periplasm.
Targeted membrane insertion. Whereas SpoIVFB and
other proteins that are synthesized in the mother-cell
region of sporulating B. subtilis cells seem to find their
septal destination by diffusion and capture, the septal
localization of the SpoIIQ protein, which is synthesized
in the forespore, is achieved by direct insertion and
capture4. SpoIIQ was found to act as a localization fac-
tor that recruits another mother-cell protein, SpoIIIAH,
REVIEWS
a B. subtilis sporulation b Diffusion and capture c Zipper model
A
Q
Forespore
FB
A Q
FB
A Q
Q lecular transport, require the assembly of scaffolds that
A by dynamic cytoskeletal filaments that adopt specific
Q overall arrangements but are highly variable on a short-
Outer forespore membrane
Figure 1 | Protein localization to the septal membrane during Bacillus subtilis
sporulation. During sporulation (a), B. subtilis undergoesNature Reviews | Microbiology
an asymmetric cell division
that generates two offspring of unequal size and developmental fate — a larger
mother cell (green) and a smaller forespore (blue). The mother cell subsequently
engulfs the forespore in a phagocytosis-like process, thereby surrounding it with a
second membrane, the so-called outer forespore membrane. During this process,
several proteins specifically localize to the interface between the two compartments
(b). In several cases, their positioning is mediated by diffusion and capture, as
exemplified by the recruitment of the freely diffusible mother cell protein SpoIVFB
(FB; purple spheres) by a pre-localized complex including the forespore protein
SpoIIQ (Q). The interaction of proteins across the mother cell and forespore
membranes involves a zipper-like mechanism (c). Using this principle, SpoIIQ engages
the mother cell factor SpoIIIA (A), which, in turn, is responsible for the septal
localization of other proteins, among them SpoIVFB.
to the forespore septal membrane5. Based on the direct
interaction between the extracytoplasmatic domains of
SpoIIQ and SpoIIIAH, a zipper-like mechanism has been
proposed as a method that is used to capture SpoIIIAH4–6
(FIG. 1c). This interaction subsequently recruits additional
factors that are synthesized by the mother cell (including
SpoIVFB)6,7, which yields a protein network that anchors
localized proteins at the septum. However, the zipper
mechanism alone is not sufficient to direct the forespore
Protofilament protein SpoIIQ to its septal position. Interestingly, it has
The basic polymeric unit of a
been shown that all of the membrane proteins that are syn-
filamentous structure; consists
of a linear row of monomers. thesized in the forespore are initially targeted to the septal
membrane, from which they slowly diffuse to the rest
Fluorescence recovery after of the forespore membrane, unless they are retained by a
photobleaching specific localization factor4. In agreement with this obser-
(FRAP). A method used to
study the dynamics of
vation, a component of the protein translocation com-
polymeric structures. A region plex, FtsY, is specifically localized to the forespore septal
within a filament that has been membrane in sporulating B. subtilis cells8. Thus, certain
assembled from fluorescently forespore proteins, such as SpoIIQ, appear to be directly
labelled monomers is bleached
targeted to their site of action by localized translocation.
by illumination with a high-
intensity laser. Subsequently, Another example of directed protein targeting is pro-
fluorescence microscopy is vided by the intracellular pathogen Shigella flexneri. To
used to monitor the kinetics facilitate movement within the host cell, this bacterium
of fluorescence recovery that positions the protein IcsA at its old cell pole, where it
results from the substitution of
bleached by unbleached
directs polymerization of host actin into a tail-like
subunits in the course of structure, thereby generating a pushing force. IcsA is
filament turnover. an outer membrane protein that has its amino-terminal
30 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
domain exposed to the host cytoplasm. During synthe-
sis, it is directly targeted to the region around the cell
pole. Concomitantly, proteolysis of IcsA occurs over the
whole-cell perimeter, in effect clearing away any protein
that has diffused away from the pole9,10. IcsA localizes
independently of its export from the cytoplasm to the
outer membrane10. It has been proposed that a region
within the amino‑terminal domain interacts with an
as-yet-unidentified factor in the polar region of the cell,
thereby targeting IcsA to its defined site of secretion
before translocation occurs11.
Stationary complexes usually have locally confined
activities or serve as regulatory landmarks. By contrast,
global processes, such as morphogenesis and macromo-
extend throughout the cell. These structures are formed
term scale. They provide force as well as directionality,
and serve as tracks for the localization of other proteins. In
the following section, we will focus specifically on protein
filaments that are involved in the determination of bacte-
rial-cell shape; other dynamic structures are discussed in
BOX 2 and in the later sections of this article.
Dynamic protein scaffolds and cell shape
A major role in subcellular organization has recently
been attributed to actin-like proteins that belong to the
MreB family. They are found in many bacteria, in which
they assemble consistently into spiral-like structures that
line the inner face of the cytoplasmic membrane. MreB
homologues play an important part in the regulation
of cell shape and have been implicated in cell polarity
and chromosome segregation12–18. Their function in
morphogenesis is dependent on the presence of another
scaffold that is formed by the extracytoplasmic protein
MreC and is modulated by accessory factors, such as the
intermediate filament protein crescentin.
The bacterial actin-like cytoskeleton. In eukaryotes, the
actin cytoskeleton consists of a highly dynamic network
of filaments, the assembly of which is regulated by the
binding and hydrolysis of ATP and is further modulated
by an abundance of accessory factors. Actin protofila-
ments possess an intrinsic polarity, whereby monomers
are preferentially added to one end and released from
the other in a phenomenon that is called treadmilling19.
Although bacterial and eukaryotic actin homologues
share minimal sequence identity (~15%), studies on
MreB from Thermotoga maritima revealed that their
three‑dimensional structures and protofilament organi-
zation are strikingly similar20,21 (FIG. 2a). Nevertheless,
the overall assembly properties of T. maritima MreB
deviate from the scheme that has been established for its
eukaryotic relatives with respect to several biochemical
parameters22.
MreB cables are highly dynamic in vivo and assume
several different architectures, comprising spiral-like
assemblies of varying curvature and length as well as
short coils, arcs and rings12,14,15,23. The mechanisms
and cellular cues that are responsible for these diverse

Box 2 | Linear arrangement of subcellular compartments — magnetosomes
There are many examples of a
intracellular membrane
compartments in bacteria,
including the chlorosomes
and chromatophores of non-
oxygenic photosynthetic
bacteria and the
magnetosomes of
magnetotactic bacteria. It is b
largely unknown how these
structures are established and
maintained at their proper
subcellular position. In the
case of magnetosomes,
however, a clearer picture of
the underlying mechanisms is starting to emerge.
Nature Reviews | Microbiology
Magnetosomes are vesicles — formed by invagination of the cytoplasmic membrane123
— that are filled with biomineralized magnetite or greigite and allow bacteria to orient
themselves in the Earth’s magnetic field124. The best-studied magnetotactic organisms,
Magnetospirillum magneticum and Magnetospirillum gryphiswaldense, contain 15–20
magnetosomes, which are arrayed in linear order parallel to the longitudinal axis of the
cell and approximately centred at mid-cell123,125. This arrangement generates a compass
needle-like structure that has an overall magnetic dipole moment that equals the sum of
the magnetic dipole moments of the individual vesicles126. As magnetosomes have an
inherent tendency to agglomerate to reduce their magnetostatic energy, the cell must
possess specific mechanisms to stabilize this linear assembly and anchor it within the cell.
Electron cryotomography analyses unveiled filamentous structures that run
alongside magnetosome chains123,125 (see the figure). The protein that is responsible
for their formation has been identified as MamK (purple in the figure), a bacterial
actin homologue that belongs to an evolutionarily distinct group of proteins that is
clearly separated from the MreB family123. It is conserved in all magnetotactic
bacteria and assembles into linear polymers that extend from one cell pole to
another123,127. In agreement with a role for MamK in magnetosome alignment,
mutants that lack the protein are still able to produce mature magnetosomes, but
fail to arrange them into a linear array. Recent work in M. gryphiswaldense has
identified a protein, MamJ, that might be involved in attaching magnetosomes to
the MamK filament (green in the figure)125. It is an acidic protein, with a repetitive
primary sequence which localizes as a linear structure that extends from pole to
pole. In a mutant strain that lacks a cluster of genes that are involved in
magnetosome biogenesis (among them mamK) this linear arrangement is abolished,
and MamJ is dispersed throughout the cytoplasm. Its cooperation with MamK in
vesicle positioning is supported by the fact that deletion of mamJ largely reproduces
the phenotype of a mamK mutant, leading to the dispersal of magnetosomes
without affecting their maturation. As a result, MamK appears to function as a track
that recruits vesicles with the help of MamJ, thereby defining the overall
orientation of the magnetosome chain. It is thought that empty and immature
vesicles are initially randomly distributed along the MamK filament (see the figure,
part a). The accumulation of magnetite (beige spheres in the figure) results in
magnetostatic interactions between individual vesicles, which leads to their
aggregation into densely packed chains (see the figure, part b) that are
subsequently stabilized by additional factors (blue in the figure )125. Figure adapted,
with permission, from Nature REF. 125  (2006) Macmillan Publishers Ltd.
patterns are unclear. In C. crescentus15,16, and its relative
Rhodobacter sphaeroides24,25, the transitions between the
different MreB structures were found to be synchro-
nized with cell-cycle progression (FIG. 2b). By contrast,
in bacteria from other phylogenetic groups, the actin-
like cytoskeleton seems to be largely unaffected by the
developmental state of the cell.
The first insights into the turnover kinetics of actin-
like filaments were provided by studies on B. subtilis.
Whereas most Gram-negative bacteria possess one
nature reviews | microbiology volume 6 | january 2008 | 31
© 2008 Nature Publishing Group
REVIEWS
mreB gene, this Gram-positive bacterium synthesizes
three actin homologues, MreB, Mbl and MreBH, which
interact with each other and assemble into a single
helical filament12,13,26,27. Fluorescence recovery after photo­
bleaching (FRAP) studies revealed a rapid exchange of
subunits within Mbl cables that occurred uniformly
and without any evidence of polarity with respect to
filament growth23. Furthermore, MreB and Mbl spirals
were observed to rotate in the cell, with one full turn
every 50–60 seconds13,26. Additional support for the
rapid turnover of MreB cables comes from a study that
analysed the movement of single MreB–GFP molecules
in live cells of C. crescentus28. The MreB subunits were
found to migrate directionally along arc-like trajectories,
which is suggestive of treadmilling of individual subunits
through stationary MreB filaments. However, the average
length of these trajectories was significantly shorter than
the MreB cable as a whole, and no common directionality
was observed relative to the overall polarity of the cell. In
C. crescentus, and possibly other bacteria, the actin-like
cytoskeleton is therefore probably composed of numer-
ous short, polarized and highly dynamic protofilaments
that associate laterally but in random orientation to each
other. The overall dynamics of this structure might be
based on the rapid exchange of protofilament bundles
rather than individual protein monomers (FIG. 2c).
Regulation of cell-wall biosynthesis by actin-like proteins.
The arrangement of actin-like proteins into large fila-
ments and their dynamic positioning within the cell are
crucial for the spatial regulation of peptidoglycan biosyn-
thesis in most rod-shaped bacteria (BOX 3). This is due, in
part, to a direct role in the positioning of the enzymes that
are involved in cell-wall formation. In B. subtilis, MreBH
was shown to interact with the peptidoglycan hydrolase
LytE27. A LytE–GFP fusion is normally found in the
form of distinct bands at the division septa and in heli-
cal patterns along the lateral cell wall. Upon deletion of
the mreBH gene, however, these lateral helical structures
are lost. As strains that lack LytE have the same defects
in cell shape as mreBH mutant cells, localization of the
hydrolase along the helical tracks of MreBH appears to
be essential for its proper function. So far, this is the only
proven example of an immediate influence of actin-like
proteins on peptidoglycan biosynthesis. In many cases,
their effect seems to be more indirect and due to a role
in the localization of MreC, a protein that is frequently
encoded in the same operon as mreB.
The role of MreC in bacterial morphogenesis. MreC
is a membrane protein that is composed of a single,
amino‑terminal transmembrane helix and a large extra-
cytoplasmic domain in Escherichia coli and B. subtilis29,30,
but is a soluble periplasmic protein in C. crescentus31.
Crystallographic data indicate that it might have the
potential to form polymeric structures30. MreC is part of
a complex that includes the poorly characterized proteins
MreD and RodA, and is essential for survival in most
bacteria that have been investigated; its inactivation
results in a loss of cell shape and subsequent lysis29,31,32.
In support of a role in cell-wall biosynthesis, MreC
REVIEWS
a Model for MreB treadmilling
(+) (–)
**
MreB–ATP
MreB–ADP
**
**
**
b MreB dynamics in C. crescentus
c Model for the architecture of MreB cables
Figure 2 | Dynamics of actin-like filaments in bacteria. MreB Nature Reviews
and | Microbiology
its orthologues
polymerize into bundles of two or more protofilaments that are arranged in parallel to
each other (a). Their assembly follows the same principles that have been observed for
actin. Polymerization occurs exclusively in the ATP-bound state (blue spheres). However,
soon after incorporation into the filament, MreB hydrolyses the nucleotide, thereby
changing into its ADP-bound conformation (purple spheres). Subunit exchange is
restricted to the ends of the filament, and the addition of new monomers occurs
preferentially at the positive-end, that is, the cap which consists of MreB subunits that are
still associated with ATP. Subunit release, by contrast, is largely restricted to the negative-
end of the filament, which is composed of MreB–ADP. In the steady state, assembly and
disassembly proceed at equal rates, resulting in the apparent sliding of individual
subunits (marked by asterisks) through the filament while the overall polymer length
remains constant — a phenomenon that is called treadmilling. In Caulobacter crescentus,
MreB cables exhibit a highly dynamic localization pattern (b). Newborn cells contain
spiral-like cables that extend between the two poles. Upon initiation of cell division,
these structures are lost and MreB condenses into a ring at the future division site. Later,
as cell constriction progresses, the zone of MreB localization gradually expands again,
leading finally to the establishment of new spirals in the two incipient daughter cells.
Biochemical evidence suggests that actin-like cables consist of numerous treadmilling
filaments that are arranged side by side in a random orientation (c).
interacts directly with the peptidoglycan synthase penicillin-
binding protein (PBP) 2, and several other high- and low-
molecular-weight PBPs (BOX 3), in C. crescentus31. Similar
results have been obtained in B. subtilis, although MreC
appears to associate preferentially with high-molecular-
weight PBPs in this organism30. These findings suggest
that the protein serves as a scaffold for the formation
32 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
of a multi-enzyme peptidoglycan biosynthetic com-
plex, thereby organizing the formation of new cell-wall
material.
The spatial arrangement of MreC is dependent on the
positioning of actin-like cables within the cell, although
the underlying mechanisms seem to vary among dif-
ferent bacteria. In E. coli, MreC interacts directly with
MreB and seems to be required for the proper assembly
of MreB into helical filaments29. Similarly, MreC forms
helical structures33 that colocalize and physically associate
with Mbl cables in B. subtilis26. The functional relevance
of this interaction is underscored by the observation that
both Mbl and MreC depletion results in the same type
of morphological defects33,34; these are caused by the
disruption of peptidoglycan biosynthesis in the longi-
tudinal parts of the cell33. In C. crescentus, an extremely
different situation is observed. Here, MreC assembles
into helical structures that do not associate with MreB
cables but, on the contrary, seem to avoid them35. The
mechanisms that underlie this effect are still unknown.
In accordance with biochemical-interaction studies31,
PBP2 was shown to colocalize with MreC in wild-type
C. crescentus cells31,35. However, the depletion of MreC
does not disrupt the already existing spiral-like structures
of PBP2, although it does specifically affect the position-
ing of newly synthesized molecules35. Thus, MreC seems
to guide peptidoglycan-synthesizing enzymes to the sites
of active cell-wall growth, where they might subsequently
be retained by interaction with other proteins or nascent
peptidoglycan strands.
Crescentin. Recent work in C. crescentus identified a
protein, named crescentin, that shares the structural
characteristics of intermediate filament proteins and acts
as a modulator of cell shape36. Although poorly conserved
on the primary-sequence level, these proteins share a
common architecture that comprises a long central
coiled-coil region that is flanked by variable head and
tail domains. Their polymerization occurs spontaneously
and does not require nucleotides or exogenous nucleation
factors37. Similar to its eukaryotic homologues, crescentin
assembles into long filaments in vitro, which illustrates
its tendency to associate laterally into small bundles36.
Localization studies revealed that the protein is positioned
specifically at the inner curvature of the crescent-shaped
C. crescentus cell. Upon inactivation of crescentin, the cells
lose their typical crescentoid morphology and grow in the
form of straight rods, which indicates that crescentin is
responsible for establishing cell curvature36. The mecha-
nism that allows this cytoskeletal structure to redirect
peptidoglycan biosynthesis remains to be established.
Morphogenetic function of the cell-division apparatus. In
addition to the systems discussed so far, the cell-division
apparatus has an important role in the regulation of cellu-
lar morphology. Not only does it comprise peptidoglycan
synthases and hydrolases that mediate the formation of
the division septum3, but, in C. crescentus, it also directs a
division-independent mode of peptidoglycan biosynthe-
sis that leads to zonal growth around the cell centre38. As
MurG, the enzyme that catalyses the last cytoplasmic step

Peptidoglycan
Meshwork of highly crosslinked
glycan strands that constitutes
the bacterial cell wall.
Penicillin-binding protein
A protein involved in
peptidoglycan biosynthesis
that is targeted and inactivated
by the antibiotic penicillin and
its derivatives.
Centromere
A region of a DNA molecule
that is attached to the
DNA-segregation apparatus.
Walker ATPase
This ATPase is characterized by
the presence of two conserved
sequence motifs (Walker A and
Walker B motif), which form
parts of the nucleotide-binding
pocket.
nature reviews | microbiology volume 6 | january 2008 | 33
REVIEWS
Box 3 | Cell-wall biogenesis
Transglycosylase
GlcNAc MurNAc GlcNAc MurNAc GlcNAc MurNAc
4 1 4 1 4 1 4 1 4 1 4 1
Transpeptidase
GlcNAc MurNAc GlcNAc MurNAc GlcNAc MurNAc
4 1 4 1 4 1 4 1 4 1 4 1
The shape of a bacterium is determined by the architecture of its cell wall (peptidoglycan) —Nature
a rigidReviews
meshwork| Microbiology
that is
formed by linear glucan strands that are crosslinked by short peptide bridges128 (see figure). Peptidoglycan biosynthesis
initiates in the cytoplasm with the assembly of a lipid-bound disaccharide–pentapeptide precursor. This basic building
block comprises the two aminosugars N‑acetylglucosamine (GlcNAc) and N‑acetylmuramic acid (MurNAc), which are
connected by a β‑1,4-glycosidic bond and a conserved five-amino-acid peptide that is attached to the lactyl group of the
MurNAc unit. Once completed, the precursor is transported across the cytoplasmic membrane and, after release from its
lipid carrier, incorporated into the peptidoglycan superstructure. This task is accomplished by a group of enzymes that are
called penicillin-binding proteins (PBPs), which include transglycosylases and transpeptidases. Transglycosylases catalyse
the formation of another β‑1,4-glycosidic bond between the disaccharide moiety of the precursor and the end of an
existing glycan strand. As a result, linear polymers of alternating GlcNAc and MurNAc units are generated, which are all
arrayed in parallel to each other. The pentapeptides, which protrude perpendicularly from the sugar backbone,
subsequently function to interconnect neighbouring glycan strands. To this end, they are linked in a pair-wise manner by
peptide bonds that are formed with the help of transpeptidases. High-molecular-weight PBPs exhibit both
transglycosylase and transpeptidase activity, whereas low-molecular-weight PBPs (such as PBP2) function as
transpeptidases only. Growth of the bacterium requires continuous remodelling of the peptidoglycan envelope. Bacteria,
therefore, also possess a series of autolytic enzymes, such as glycosidases, peptidases and amidases, that selectively
cleave the molecular meshwork and thereby facilitate the insertion of additional cell-wall material.
of peptidoglycan formation, was found to be associated Plasmid segregation. The first indication that replicated
with the cell-division apparatus throughout the period DNA might be inherited in a non-random way came
of zonal growth38,39, medial cell-wall extension might be from work on low-copy-number replicons. Localization
promoted by restricting the delivery of peptidoglycan studies revealed that the copies of these plasmids are
precursors to the mid-cell region. However, future studies actively separated from each other and positioned in
are necessary to determine whether this process is addi- the incipient daughter-cell compartments before cell
tionally supported by the recruitment of a specific set of division occurs41–46. In all cases that have been inves-
peptidoglycan synthases from the periphery to the cell tigated in detail, partitioning relies on the activities of
centre. MurG is also enriched at the cell-division site of three different plasmid-encoded factors — a centro-
E. coli40, which suggests that mid-cell growth zones could meric sequence, a centromere-binding protein and an
be a common phenomenon in bacteria. ATPase that interacts with the centromeric nucleopro-
tein complex. Despite this common theme, the nature
Bacterial DNA segregation of the individual factors and the segregation apparatus
Evidence has accumulated that, in addition to protein varies considerably among different plasmids. The most
complexes, plasmids and chromosomes are also actively important difference concerns the ATPase component,
positioned within bacterial cells. As for morphogenesis, which can be either a Walker ATPase (type I partitioning
dynamic cytoskeletal filaments have emerged as essential system), a member of the actin superfamily (type II plas-
factors in the underlying localization mechanisms. mid-partitioning system) or a tubulin homologue.
© 2008 Nature Publishing Group
REVIEWS

a b
ParM–ATP

Assembly ParM–ADP

ParR

GTP hydrolysis

Catastrophe Stabilization

Figure 3 | Plasmid segregation by the actin homologue ParM. Unlike actin polymers, which eventually reach a steady
Nature Reviews
state that is characterized by an equilibrium between subunit addition and dissociation (treadmilling), | Microbiology
ParM filaments
continuously cycle between phases of rapid growth and complete disassembly (a). Biochemical analyses showed that
only the ATP-bound form of ParM (blue spheres) is able to polymerize efficiently. However, owing to the cooperative nature
of its ATPase activity, ParM rapidly hydrolyses its bound nucleotide once it has become integrated into a filament
(purple spheres). Nevertheless, the overall structure remains stable and continues to grow as long as its ends remain
capped by ATP-bound subunits. Once these are lost, for example, owing to a shortage in the supply of ParM monomers, the
filament starts to depolymerize rapidly (catastrophe). Stabilization of the caps, by contrast, prevents disassembly and
allows the polymer to continue elongation. ParR nucleoprotein complexes recruit filaments that have formed
spontaneously in the cytoplasm and promote their extension into long polymeric structures. As a consequence, plasmids
are pushed apart and moved to opposite ends of the cell (b).

Walker A cytoskeletal
ATPases
(WACA). A group of Walker
ATPases that possess a distinct
version of the Walker A motif
that deviates from the universal
consensus. These proteins
share structural similarity with
P‑loop GTPases and are
recognized as members of the
GTPase superfamily.
Plasmid segregation by actin-like proteins. The best-
studied partitioning system that is based on an actin-like
ATPase is that of the conjugative resistance plasmid R1.
Its centromeric region (parC) is bound by the DNA-
binding protein ParR47. This complex is then recognized
by ParM, an actin-like protein, the ATPase activity of
which is essential for plasmid stabilization48,49.
In vitro, ParM polymerizes in an ATP-dependent
manner, forming filaments that are reminiscent of
F‑actin50,51. Despite this similarity, its assembly kinetics
have several distinct features. For example, the nuclea-
tion of ParM is a rapid and spontaneous process51,52,
with filaments elongating in a symmetrical bidirectional
fashion that is contrary to the polarized growth of actin52.
In addition, ParM filaments never reach an equilibrium
between association and dissociation, as is typical for
treadmilling actin polymers, but instead continuously
cycle between phases of rapid growth and complete dis-
assembly52,53 (FIG. 3a). This behaviour, which is known as
dynamic instability, has previously only been observed
for eukaryotic tubulin54.
Localization studies have revealed that ParM can
form axial filaments in E. coli51. Each filament is flanked
by two copies of plasmid R1 that appear to be pushed
apart towards the cell poles by the polymerization of
ParM between them48. The biochemical mechanism
of this segregation process has recently been unravelled
by reconstituting the R1 partitioning system in vitro53.
When a DNA fragment that contained the R1 parS
region was attached to beads and mixed with ParR and
ParM, the addition of ATP induced the formation of
numerous short filaments that extended from the bead
surface and, owing to their dynamic instability, went
through cycles of rapid growth and shrinkage. However,
as soon as two of these beads came into proximity, the
filaments between them suddenly started to grow and
push them apart. Elongation occurred exclusively at the
interface between ParM and the centromeric nucleo-
protein complex and was based on the stabilizing effect
of the ParR–parS complex on the filament ends, which
prevented the spontaneous disassembly of the polymer.
These data suggest that the three-component partition-
ing system of R1 is sufficient to place plasmid copies at
opposite cell poles without the help of additional host
factors (FIG. 3b).
Interestingly, the B. subtilis plasmid pBET131 was
found to encode a novel actin-like partitioning ATPase
named AlfA that lacks dynamic instability and has polym-
erization kinetics that are similar to those of MreB55. Thus,
actin homologues have adopted different molecular
mechanisms to mediate the movement of sister plasmids
into the two daughter-cell compartments.
Plasmid segregation by Walker-type ATPases. Most
non-actin-based partitioning ATPases are members of
the so-called Walker A cytoskeletal ATPase (WACA) fam-
ily (type I partitioning systems)56. Analogous to the R1
partitioning system, each of these factors interacts with
a DNA-binding protein that associates cooperatively
with a cluster of conserved sites on its cognate plasmid.
Despite differences in their primary sequences, overall,
WACA ATPases show similar behaviour in vitro. They
possess weak cooperative ATPase activity and are capable
of polymerizing in an ATP-dependent manner, forming

34 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group

Nucleoid
A distinct region within the
cytoplasm that harbours
the chromosomal DNA.
Origin of replication
A chromosomal site that serves
as the starting point of the
bidirectional DNA-replication
process.
Terminus
A chromosomal region in which
the two replication forks meet
towards the end of DNA
replication.
nature reviews | microbiology volume 6 | january 2008 | 35
massive protofilament bundles that measure up to sev-
eral micrometres in length. However, detailed analyses of
their polymerization dynamics and interactions with the
centromeric nucleoprotein complex have not yet been
performed. The biology of these systems has recently
been described in several excellent reviews57–59.
Although type I and type II partitioning systems
share some common principles, their modes of action are
fundamentally different. Whereas ParM assembles into
stable axial filaments that push plasmids to opposite cell
poles48,53, WACA ATPases exhibit a highly dynamic locali-
zation pattern in vivo. SopA and ParA, which are encoded
by the E. coli plasmids F and pB171, respectively, were
shown to oscillate back and forth across the nucleoid at
~20 minute intervals60,61. However, the molecular mecha-
nisms that couple the dynamic assembly of partitioning
proteins to plasmid stabilization remain to be elucidated.
Studies on the localization of plasmids F and pB171
revealed that at the beginning of their division cycle most
cells contain a single plasmid cluster that is positioned at
the cell centre. Later on, this cluster is duplicated, and the
newly generated copies are moved to the quarter positions
of the cell. Cytokinesis then occurs halfway between the
two clusters, resulting in daughter cells that each carry
a single cluster at the mid-cell42,43,45. If the copy number
of pB171 is increased artificially, cells accumulate up to
seven plasmid clusters that are evenly distributed along
the long axis of the cell62. This finding indicates that type I
partitioning systems do not move plasmids to defined
subcellular positions, as is the case for the R1 partition-
ing system, but rather distribute them to clusters that are
arranged in regular arrays within the nucleoid.
Interestingly, the cytoplasmic chemotaxis sensory
clusters of R. sphaeroides have a localization pattern that
is strikingly similar to that of plasmids that are segregated
by type I partitioning systems. The proper positioning
of these clusters was found to be dependent on a pro-
tein, called PpfA, that is homologous to WACA plasmid
partitioning ATPases63, which suggests that bacteria can
use similar mechanisms to arrange proteins and DNA
within the cell.
Plasmid segregation by a tubulin homologue. A third
class of plasmid-segregation systems is based on the
activity of tubulin homologues. Recent work showed
that plasmid pBtoxis from Bacillus thuringiensis serovar
israelensis encodes a protein — designated TubZ — that
shows significant similarity to tubulin64,65 and is essential
for its stable partitioning. TubZ assembles into highly
dynamic filaments that translocate rapidly through the
cell65. FRAP analyses revealed that filament migration
is achieved by an actin-like treadmilling mechanism.
However, it is unclear how the translocation of TubZ
filaments is related to plasmid stabilization.
The unusual characteristics of TubZ, a tubulin homo-
logue with actin-like dynamics, and ParM, an actin
homologue with tubulin-like dynamics, demonstrate
that bacterial and eukaryotic cytoskeletal proteins have
diverged considerably with respect to their function and
polymerization mechanisms, even though they share the
same evolutionary origin and overall structures.
© 2008 Nature Publishing Group
REVIEWS
a ori
ter
b
ori
ter ori
ter
C. crescentus E. coli
Figure 4 | The arrangement of chromosomal DNA in
bacteria. Consecutive segments of chromosomal DNA are
Nature Reviews | Microbiology
folded into supercoiled domains that are stacked on top of
each other and arranged into a circular superstructure (a).
Consequently, the subcellular position of a locus correlates
directly with its location on the circular chromosomal map.
In Caulobacter crescentus, the origin of replication (ori) is
localized to the flagellated pole of the swarmer cell,
whereas the terminus (ter) is found at the opposite pole (b).
Therefore, the left and right arms of the chromosome are
oriented in parallel to the long axis of the cell. In
Escherichia coli, by contrast, ori and ter are both located at
mid-cell, such that the two arms of the chromosome flank
the transverse cellular axis. Panel a adapted, with
permission, from REF. 77  (2006) Elsevier Science.
Arrangement of chromosomal DNA. Bacterial genomes
are usually organized into circular chromosomes (one or
several) that are up to several megabase pairs in size. It has
long been unclear how the cell packages these enormous
molecules into the confined space of its cytoplasm and
concomitantly ensures their faithful replication and par-
titioning during each cell-division cycle. With the advent
of site-specific labelling methods, it has become possible
to probe the arrangement of chromatin in a systematic
fashion66,67. An early study in B. subtilis determined the
subcellular location of four chromosomal loci and found
that their arrangement followed a reproducible pattern68.
Conclusive evidence that chromosomal DNA is not dis-
tributed randomly within the cell but in fact has a highly
conserved organization was provided by a large-scale
analysis that investigated the subcellular positioning of
112 chromosomal loci in C. crescentus69 (FIG. 4). This work
showed that the origin of replication is invariantly found
at the flagellated pole of the bacterium and the terminus
is located at the opposite end of the cell. Other loci are
arranged between these two fixed points in exactly the
same order as on the circular chromosomal map. A simi-
lar correlation between the subcellular and chromosomal
positions of loci was revealed in E. coli. However, unlike
C. crescentus, E. coli places the origin and terminus regions
at the cell centre, and the two arms of the chromosome
(replichores) form separate domains that are located on
opposite sides of its transverse axis70,71. The chromosomal
REVIEWS
architecture of other organisms has not been analysed in
detail yet, but it is likely that the linear ordering of loci is
a common principle among bacteria.
So, how is this conserved arrangement established?
Studies on the movement of newly duplicated chromo-
somal regions showed that the specific localization of each
site is established during the DNA-segregation process.
Immediately after the initiation of replication, the newly
synthesized origin regions are separated and rapidly
moved to their conserved positions in the incipient
daughter cells69,72–75. Subsequently, the remaining bulk of
the chromosome is duplicated progressively from the ori-
gin to the terminus region. Similar to the origin regions,
the two new copies of each locus are quickly partitioned.
They are condensed immediately after emergence from
the replisome, segregated into the daughter-cell com-
partments and added successively to the newly replicated
DNA that has already been deposited there69,70,76. Unlike
eukaryotes, therefore, bacteria segregate their DNA while
replication is in progress, with the two sister nucleoids
growing in layers that reflect the succession of loci on
the chromosome. The mechanisms that mediate bacte-
rial-chromosome dynamics are unclear, and several dif-
ferent models have been proposed to account for the rapid
and ordered movement of loci during the chromosome-
segregation process77. The current understanding is that
the two copies of the origin are actively separated from
each other and positioned in the incipient daughter cells,
so that they serve as landmarks for the establishment of the
new sister nucleoids. The bulk of the chromosome then
follows as a consequence of DNA condensation, probably
supported by the activities of other factors. Finally, the two
sister chromosomes are decatenated and actively cleared
from the cell-division site to allow cytokinesis to occur78.
Chromosome segregation. The existence of an active
mechanism that mediates origin segregation is supported
by the fact that, in all bacteria investigated, movement
of the two origin copies occurs in a highly reproducible
and directed manner and is significantly faster than cell
elongation69,79,80. One of the factors that has been impli-
cated in this partitioning process is the cytoskeletal protein
MreB. Its overproduction, depletion16,34,81 or inactivation
by the antibiotic A22 (Refs 17,18) results in misplacement
of the origin regions, and chromosome-segregation defects
in various bacteria. Moreover, MreB was shown to inter-
act, directly or indirectly, with a chromosomal region that
flanks the origin of replication in C. crescentus17. However,
it has remained controversial whether these results actually
reflect a direct involvement in DNA segregation.
Other studies have provided evidence of a role for
type I partitioning systems in chromosome segrega-
tion. In most bacteria, an operon encoding the WACA
ATPase ParA and its cognate centromere-binding protein
ParB is located in the vicinity of the replication origin,
whereas conserved ParB-binding sites (parS) are scattered
throughout the origin-proximal region of the chromo-
some82. Recent studies in B. subtilis demonstrated that,
upon binding to individual sites83, ParB spreads into the
flanking chromosomal regions, forming nucleoprotein
complexes that cover up to 20 kilobases (kb) of DNA84,85.
36 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
These individual complexes then aggregate into a single
centromere-like superstructure in a ParA-dependent
manner86. Chromosomally encoded partitioning systems
are able to functionally replace plasmid-borne type I
partitioning systems and confer stability to intrinsi-
cally unstable plasmids, indicating that they indeed
constitute active segregation machineries87,88. In agree-
ment with this finding, the ParAB system was shown
to be required for proper transfer of the chromosomal
origin region into the forespore compartment at the onset
of sporulation in B. subtilis89. In doing so, it cooperates with
a sporulation-specific protein, named RacA, that serves to
tether the origin-proximal region of the chromosome to
the cell pole89–91. Moreover, vegetative cells of B. subtilis
that are deficient in ParA or ParB fail to separate their
origin regions after duplication, although the bulk of the
chromosome is left unaffected92.
Evidence for a direct role of ParAB in origin seg-
regation has come from the study of Vibrio cholerae, a
bacterium that possesses two circular chromosomes, of
which each encodes its own type I partitioning system.
The smaller chromosome (ChrII) has the typical segre-
gation pattern that is shared by low-copy-number plas-
mids, such as F and pB171, and the larger chromosome
(ChrI) exhibits dynamics that are similar to those of the
C. crescentus chromosome80,93. Interestingly, ParBI, which
binds to 3 parS sites that are approximately 65 kb to the
right of the replication origin of the large chromosome,
is consistently localized closer to the cell poles than to
the origin itself and moves ahead of the origin during
the segregation process94. These findings suggest that
the ParBI–parS complex defines the chromosomal cen-
tromere and mediates segregation and polar positioning
of the origin regions. ParBI and the origin regions lose
their polar localization in mutants that lack ParAI and
are instead found near the cell centre before the start of
S phase and around the quarter positions in cells that
have initiated DNA replication. Therefore, ParAI seems
to be necessary for correct positioning of the ParBI–parS
complex. Time-lapse analyses indeed showed that the
protein assembles into a dynamic polymeric structure
that seems to pull the moving ParB I–parS complex
from the old pole towards the new pole94. In doing
so, the ParAI structure first stretches throughout the
cell and then progressively retracts in the direction of
the new pole, with its lagging edge colocalizing with
the moving ParBI–parS complex. Thus, a mitotic-like
mechanism that is based on type I partitioning systems
seems to mediate origin segregation in V. cholerae and,
presumably, other bacteria.
V. cholerae cells that lack ParAI only have minor
growth and overall chromosome-partitioning defects94,
and similar observations have been made for most other
bacteria in which the type I chromosome-partitioning
systems had been inactivated82. These findings indicate
that active segregation of the newly synthesized origin
regions is not necessarily required for the establish-
ment of two fully separated sister nucleoids. As a result,
chromosome segregation might rely, in part, on other
mechanisms, the precise nature of which remains to be
determined.

a E. coli b C. crescentus
MinE
MipZ
MinD FtsZ Nucleoid
Origin
FtsZ
ParB
MinC Release of MinCD Origin segregation
Assembly of a new patch Displacement of FtsZ
Start of the next cycle Z-ring formation
Figure 5 | Positioning of the cell-division plane. In Escherichia
Naturecoli, assembly
Reviews of the FtsZ
| Microbiology
ring is restricted to the mid-cell by nucleoid occlusion and the MinE-driven pole-to-pole
oscillation of the cell-division inhibitor MinCD (a). By contrast, division-plane localization
in Caulobacter crescentus is mediated by MipZ, an inhibitor of FtsZ polymerization that
forms a complex with the DNA-binding protein ParB at the chromosomal origin of
replication, thus exploiting chromosome dynamics for its subcellular positioning (b).
Panel b adapted, with permission, from REF. 113  (2006) Elsevier Science.
To generate offspring that contain equivalent DNA,
the cell must not only provide a mechanism for proper
chromosome segregation, but also ensure that cytokinesis
occurs precisely between the two newly formed sister
nucleoids. The core machinery that mediates cell divi-
sion is conserved in most bacteria. Nevertheless, several
independent mechanisms have evolved to determine the
subcellular site of its assembly.
Division-site placement
Formation of the bacterial-cell-division apparatus, called
the divisome, occurs in a multistep process that is initi-
ated by assembly of the tubulin homologue FtsZ into a
ring-shaped structure at the future division site. This
cytoskeletal element mediates the recruitment of all other
divisome components, both directly and indirectly, and
plays a crucial part in the subsequent constriction proc-
ess3. Although the three-dimensional structures of FtsZ
and tubulin are remarkably similar95, the two proteins
show distinct polymerization behaviour. The assembly
dynamics of FtsZ have been discussed extensively in
several recent reviews56,96,97. Most importantly, FtsZ has
a high propensity to polymerize in vivo, forming spiral-
and arc-like structures that are in a constant process of
polymerization and disassembly. Their consolidation at
Nucleoid occlusion
The inhibitory effect of the
the cell centre, which results from the destabilizing effect
nucleoid on the formation of of negative regulators in the polar regions of the cell, is
the septal FtsZ ring. thought to provide the basis of septal-ring formation.
nature reviews | microbiology volume 6 | january 2008 | 37
© 2008 Nature Publishing Group
REVIEWS
The Min system. The first mechanism that was shown to
control placement of the bacterial division site was dis-
covered in E. coli. It is based on the activity of three pro-
teins, encoded by the minCDE operon, that cooperate to
establish a fascinating, self-contained oscillatory system98
(FIG. 5a). MinD is an ATPase of the WACA family and
interacts with MinC to form a membrane-associated, top-
ologically unspecific inhibitor of FtsZ-ring formation99,100.
Owing to the activity of MinE, however, the MinCD com-
plex is normally restricted to the polar regions of the cell,
thus only allowing cell division to occur close to the cell
centre101,102. MinCD continuously oscillates between the
two cell halves in 40–50 second intervals103–105. In doing
so, it first assembles into a large polymeric patch at one
end of the cell. Subsequently, this cap-like structure starts
to shrink, gradually losing subunits from the pole-distal
end, until it has completely disappeared. Concomitantly,
a new MinCD assembly forms at the opposite pole, and
the cycle starts again. MinE forms a circular structure that
follows the retracting edge of the MinCD patches101,106. In
its absence, the oscillatory behaviour of the system is lost,
and the MinCD complex is evenly dispersed throughout
the membrane103. This mechanism is widely used among
bacteria, albeit frequently in modified forms. B. subtilis,
for example, lacks MinE and positions the MinCD com-
plex statically at its cell poles107,108. Details on the molecular
function of the Min proteins are provided in two recent
comprehensive reviews96,109.
Nucleoid occlusion. In mutants that lack a functional
Min system, cell division occurs either close to the poles
or between nucleoids, but never on top of regions that
contain chromosomal DNA110. Recent studies identi-
fied two unrelated proteins, Noc from B. subtilis111 and
SlmA from E. coli112, that mediate this so-called nucleoid
occlusion effect (FIG. 5a). In their absence, Min-deficient
cells accumulate numerous FtsZ clusters that are ran-
domly distributed throughout the cytoplasm. These
clusters frequently overlap with the nucleoids and, under
certain conditions, promote cell-division events that lead
to dissection of the chromosome. However, the inactiva-
tion of NocA or SlmA has little effect on FtsZ localization
in cells that have a functional Min system, which suggests
that nucleoid occlusion might serve as a fail-safe mecha-
nism that ensures proper cell division under conditions
of unbalanced growth. However, how do Noc and SlmA
function? Both proteins possess a helix–turn–helix DNA-
binding motif that allows them to interact nonspecifically
with chromosomal DNA and therefore to colocalize with
the nucleoid. SlmA was shown to mediate the recruitment
of FtsZ to the nucleoid if expressed at high levels and has
a strong bundling effect on FtsZ filaments in vitro112.
Nucleoid occlusion factors might, therefore, function by
out-competing other cell-division proteins that bind to
and stabilize the FtsZ ring, thereby efficiently preventing
the assembly of a functional divisome.
Regulation of cell division by MipZ. There are several
organisms that lack MinCDE homologues and nucle-
oid occlusion but yet still manage to divide properly in
the mid-cell region, which suggests the existence of
REVIEWS

alternative regulatory mechanisms. Recent work in
C. crescentus has indeed identified a novel system that
couples the assembly and positioning of the FtsZ ring to
the initiation of chromosome replication and the bipolar
positioning of the duplicated origin regions113 (FIG. 5b).
Its central component is MipZ, an essential ATPase
that has weak similarity to ParA-like DNA-partitioning
proteins and is widely conserved among alphaproteo-
bacteria. MipZ directly interacts with the chromosome-
partitioning protein ParB113, which, in turn, binds to a
cluster of sites (parS) that are approximately 15 kb away
from the chromosomal origin of replication114,115. Together
with the origin region, the resulting complex is positioned
at the old pole in newborn cells113. Initiation of DNA rep-
lication then generates two copies of the parS-containing
segment, both of which are immediately decorated with
ParB and MipZ. During the subsequent segregation proc-
ess, one of these segments stays at the original position,
while the second copy moves rapidly across the cell to the
opposite pole. MipZ interacts with ParB in a highly dynamic
manner that is regulated by ATP binding and hydrolysis113.
Consequently, it is not stably tethered to the origin regions
but rather is distributed in a gradient, with its concentra-
tion progressively increasing towards the polar ParB–parS
complexes. In vitro studies demonstrated that MipZ acts as
an inhibitor of FtsZ polymerization113. Consequently, FtsZ
is consistently found in the subcellular region that exhib-
its the lowest concentration of MipZ. In newborn cells, it
initially forms a focus at the new pole, opposite the
single MipZ–ParB complex that is located at the old pole.
After duplication and segregation of the origin regions,
the polar FtsZ aggregate is disassembled and a new one
is formed at the cell centre. Synthesis of the other cell-
division proteins, which occurs later in the C. crescentus
cell cycle116, might then stabilize FtsZ into a septal ring
that can establish a functional divisome and initiate
cytokinesis. This mechanism ensures that the division site
is positioned between the two nascent sister nucleoids.
Based on its homology to ParA-type WACA ATPases, and
its interaction with ParB, MipZ is probably derived from
a plasmid-partitioning protein that later adopted a role in
the spatial regulation of cell division.
Concluding remarks
Flexibility in the use of dynamic protein structures is
a common theme in bacteria. Tubulin filaments are
elementary parts of the cell-division apparatus as well as
mediators of plasmid segregation. Similarly, actin cables
function in DNA partitioning, cell-shape determination
and protein and vesicle localization. Comparable diversity
is also observed for WACA ATPases, which form filaments
that are involved in DNA segregation as well as dynamic
regulatory complexes that define the cell-division plane.
This astonishing range of mechanisms has been identi-
fied almost exclusively in a handful of established model
organisms that represent only a small fraction of the bac-
terial world. Therefore, novel systems that are involved in
the dynamic temporal and spatial organization of bacteria
might await discovery in the future.
Although several different systems that mediate the
spatial organization of bacteria have been identified, their
function is often poorly understood. Notably, the factors
that mediate the specific positioning of protein complexes
at the bacterial cell poles remain obscure. Recent work in
C. crescentus identified a membrane protein, TipN, that
interacts with the cell-division apparatus and remains
attached to the new poles after cytokinesis, so serving as
a landmark that regulates the assembly and positioning
of the flagellum in the two daughter cells117,118. However,
even though this system provides an elegant way to
pass on positional information to the next generation
and to differentiate between the old and new cell pole,
the primary determinants that retain TipN at its polar
position are still unknown. One of the structures that
might help to define the cell poles is peptidoglycan. Its
architecture at the curved polar regions is probably differ-
ent from that in the straight longitudinal sections of the
cell, thus providing a specific target for peptidoglycan-
binding proteins. In addition, there are data that
suggest a role for cardiolipin in polar protein localiza-
tion. This minor lipid species was reported to accumu-
late, possibly owing to a self-organizing process119, in the
polar regions of the cytoplasmic membrane in E. coli120
and B. subtilis121, and it has been suggested that it medi-
ates polar positioning of the osmosensory transporter
ProP in E. coli122. However, although both the peptidog-
lycan- and cardiolipin-mediated localization mecha-
nisms are appealing, future work is required to test their
validity.
Aside from polar targeting, little information is avail-
able on the biochemical mechanisms that are responsible
for the assembly of bacterial actin homologues, tubulin
homologues and WACA ATPases into dynamic cytoskele-
tal filaments. The same applies to the temporal and spatial
regulation of these structures as well as their interplay with
other cellular factors. Finally, further studies are required
to fully understand the molecular details that underlie the
regulation of division-site placement by nucleoid occlu-
sion and MipZ. As demonstrated by FtsZ positioning in
C. crescentus, different spatial regulatory systems can be
tightly interconnected. It will, therefore, be a major task
to elucidate the interdependence of dynamic processes in
the cell and merge the knowledge of individual processes
into a global concept of cellular function.

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Acknowledgements
This work was supported by funding from the Max Planck
Society to M.T. and grants from the National Institutes of
Health to L.S.
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bacillus subtilis | Caulobacter crescentus | Escherichia coli
Magnetospirillum gryphiswaldense | Magnetospirillum
magneticum | Rhodobacter sphaeroides | Shigella flexneri |
Thermotoga maritima | Vibrio cholerae
Entrez Protein:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein
FtsY | IcsA | MreC | SpoIVFB | SpoIIQ | SpoIIIAH | TipN
FURTHER INFORMATION
Martin Thanbichler’s homepage: http://www.mpi-marburg.
mpg.de/thanbichler/
All links are active in the online pdf
 REVIEWS

Drugs versus bugs: in pursuit of the

persistent predator Mycobacterium
tuberculosis
James C. Sacchettini*, Eric J. Rubin‡ and Joel S. Freundlich*
Abstract | Tuberculosis (TB) claims a life every 10 seconds and global mortality rates are
increasing despite the use of chemotherapy. But why have we not progressed towards the
eradication of the disease? There is no simple answer, although apathy, politics, poverty and
our inability to fight the chronic infection have all contributed. Drug resistance and HIV-1
are also greatly influencing the current TB battle plans, as our understanding of their
complicity grows. In this Review, recent efforts to fight TB will be described, specifically
focusing on how drug discovery could combat the resistance and persistence that make TB
worthy of the moniker ‘The Great White Plague’.

*Department of Biochemistry
and Biophysics, Texas A&M
University, College Station,
Texas 77843, USA.
‡
Department of Immunology
and Infectious Diseases,
Harvard School of Public
Health, Boston,
Massachusetts 02115, USA.
Correspondence to J.C.S.
e‑mail: sacchett@tamu.edu
doi:10.1038/nrmicro1816
Many think of tuberculosis (TB) as the scourge that
devastated Europe in the seventeenth century and
rapidly became a leading cause of death worldwide
before being virtually eliminated (BOX 1). TB has been
brought back to our thoughts, however, by the recent
reports of outbreaks of drug-resistant disease. In fact,
TB never ‘went away’, but has remained the ‘Captain
of Death’ throughout much of Asia and Africa. TB
mortality rates are once again on the rise — this has
been attributed to the HIV-1 epidemic, which has pro-
duced a new and highly susceptible population, and the
inconsistent use of antibiotics, which has led to a new
epidemic of drug-resistant disease in many parts of the
world. Given that investment in antibiotics in general,
including antitubercular drugs, has waned over recent
years, we have found ourselves unable to respond to the
resurgence of TB.
Two factors, persistence (BOX 2) and resistance,
have made the treatment of the causative organism,
Mycobacterium tuberculosis, difficult. The term persist-
ence describes the survival of M. tuberculosis despite the
use of antibiotics (rather than latency, which refers to
the ability of apparently dormant bacteria in asympto-
matic infected individuals to activate as much as decades
after the initial infection). Little concrete information is
available on the cellular or metabolic status of persistent
mycobacteria. As a consequence of persistence, drug
treatment is extended, and current antibiotics (BOX 3)
require long courses of treatment to cure patients and
prevent relapse. Currently, even the most effective regi-
mens require a combination of at least 3 drugs and last
for 6 months. As patients feel better within 1–2 weeks,
they have little motivation to complete therapy. Thus,
current World Health Organization guidelines call for
treatment to be directly observed. The required infra-
structure for drug-delivery and treatment supervision
can be difficult to provide in much of the world, particu-
larly in areas that are afflicted with poverty and unstable
governments.
For TB, drug resistance is due to genetic muta-
tions that result in a heritable loss of susceptibility to
antibiotics. These mutations generally occur either in
the target or the activator of the drug. TABLEs 1,2 sum-
marize the most common mutations for current first-
and second-line drugs, and refer to their respective
mechanism (or mechanisms) of action and half-lives.
Slow-acting drugs, combined with poor health-care
systems, have led to incomplete treatment, relapse
and the emergence of resistant mycobacteria. Strains
of M. tuberculosis that are resistant to at least one of
the first-line antibiotics (TABLE 1) have become com-
mon, and strains that are resistant to two or more are
not uncommon. Although resistance to single agents
generally still permits cure (albeit by using even more
extended courses of therapy), strains that are resistant
to multiple agents cause disease that is far more dif-
ficult and costly to treat. This is particularly true for
multidrug-resistant (MDR) strains that are resistant
to the first-line drugs isoniazid (INH) and rifampicin.
Some strains carry far greater levels of drug resistance,
including extensively drug-resistant (XDR) bacteria,
which are MDR and also resistant to fluoroquinolones

nature reviews | microbiology volume 6 | january 2008 | 41

© 2008 Nature Publishing Group
REVIEWS

Box 1 | Waksman’s vision

In his speech at the Nobel Banquet in December 1952, Selman Waksman said “The Great White Plague, which only
10 years ago was thought to be immune to drug therapy, is gradually being eliminated…streptomycin pointed a way.
Later supplemented with PAS and more recently with isoniazid, it has brought the control of this disease within sight.”
In 1943, Selman Waksman, a microbiologist at Rutgers University, purified a compound from the soil bacterium
Streptomyces griseus that could kill a wide spectrum of bacteria, including Mycobacterium tuberculosis, in culture and in
animals125,126. Unlike the discovery of penicillin 15 years earlier, Waksman’s discovery of streptomycin was not accidental.
His student, Albert Schatz, conducted a directed screen of 10,000 cultures of soil bacteria to identify those that inhibited
the growth of co-cultured Gram-negative bacteria. He found only ten cultures that could significantly block the growth
of the test bacteria. The Nobel-Prize-winning discovery was heralded by most as the ‘beginning of the end for
tuberculosis (TB)’, although the prize itself has been controversial as some have argued that it should have been shared
with his student, or with Jorgen Lehmen, who discovered the TB drug para-aminosalicylic acid (PAS) in the same year as
the discovery of streptomycin127. The drug was quickly approved by the United States Food and Drug Administration, and
within 2 years Merck was producing 25,000 kg per day of streptomycin. Although the discovery of streptomycin proved
that a bacterium was the cause of the disease and led to almost miraculous responses in patients infected with TB, it
required repeated injection and was associated with significant toxicity. Worse, strains of M. tuberculosis that were
resistant to the drug were discovered within a few months of use.

and at least one injectable antibiotic. These infections
are extraordinarily difficult to treat using the current
agents.
Clearly, our current drug armamentarium (TABLES 1,2)
has not been sufficient to control the TB epidemic
(BOX 4) . New antibiotics, particularly those that are
derived from new chemical classes, are more likely to
have activity against many drug-resistant strains. The
path to creating antibiotics that act against persistent
organisms and produce more rapid clearance of infec-
tions is less clear. Certainly, a vital part of drug develop-
ment is the understanding of the physiology of growing
and persistent organisms, an area in which little infor-
mation is available, as reviewed elsewhere1–3. A greater
knowledge of persistence could lead to a directed strat-
egy for the development of more rapidly effective anti-
biotics. However, until a clear picture of persistence and
its role in infection is achieved, researchers will have to
rely on more empirical approaches, as demonstrated by
the discovery of TMC207 (discussed below).
Here, we review recent and ongoing efforts to pro-
duce new antitubercular drugs, and the properties of
current investigational agents. This Review seeks to
complement other recent discussions of TB research,
such as those by Janin4, Ginsberg and Spigelman5, and
Williams and Duncan6. We will follow a ‘drug timeline’,
beginning with a discussion of discovery technologies
that were designed to provide new clinical antitubercular
candidates, before considering compounds that are cur-
rently in trials and, finally, already approved drugs that
are sought to be used as TB therapies.
Drug-discovery methods
Genetic approaches to target identification. Ideal TB
drug targets should have three characteristics: they
should be required for bacterial growth and persistence
(that is, they must be expressed and essential during
the time that treatment occurs); it should be possible to
inhibit their activity using small molecules (that is, the
target should be ‘druggable’)7,8; and they should be acces-
sible to these modulatory compounds. Theoretically, the
simplest way to find targets that have these ideal char-
acteristics is to discover an active compound and then
define its target. In practice, however, this has not been
so simple, and we still do not know the targets of many
existing antitubercular drugs that are in clinical use.
Recently, two systematic approaches have emerged
to define the targets of compounds that have activity
against M. tuberculosis. Expression analysis provides a
method to profile cellular responses to perturbations,
such as small molecules or environmental stress. Several
groups have collected a large number of datasets from
DNA microarray experiments that were performed
under various conditions. In particular, Boshoff and
colleagues9 have evaluated bacterial gene expression in
response to a number of drugs, toxins and environmental
conditions. Although these results do not define a single
molecular target, they can be used to identify susceptible
pathways that may contain drug targets.
The recent availability of affordable whole-genome
sequencing also potentially provides a rapid way of
finding targets. For example, Andries and colleagues10
selected for mutants that were resistant to the drug
TMC207 (discussed below) and sequenced their entire
chromosomes. All resilient strains contained mutations
in a single gene that encoded a subunit of ATP synthase.
However, this success story is not always easily replicated.
Box 2 | Bacterial persistence and treatment failure
Why does antibiotic treatment fail even when bacteria are
not genetically drug resistant? This phenomenon, often
termed bacterial ‘persistence’, might be explained in a
number of ways, given that Mycobacterium tuberculosis
induces a chronic inflammatory response in which
bacteria are sequestered from drugs in tissue. The local
concentration of antibiotics in lesions, such as granulomas,
might not be adequate to cause bacterial death, or some
bacteria might adopt a physiological state that renders
them less susceptible to antibiotics. This could be a
stochastic process, in which a subpopulation of cells adopt
a physiological state that renders them drug insensitive.
Alternatively, an environmental condition, such as low
oxygen or carbon starvation, might induce the persistent
state.  All of these hypotheses could be true. However, as
yet, we do not know if any of them have an important role
in treatment failure.

42 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group
 REVIEWS

Box 3 | Lessons from current antitubercular drugs

There is an excellent chance that patients who have tuberculosis (TB) can be cured using currently available drugs if
they are able to complete the required course of therapy. But what characteristics should new drugs have to improve
on current treatment?
Oral bioavailability
• Streptomycin is a highly potent drug but is rarely used, partly because it must be injected.
Good tolerance
• Para-aminosalicylic acid (PAS) was largely abandoned early on because of gastrointestinal intolerance.
Usability in multiple populations
• Fluoroquinolones are not recommended for the treatment of pregnant women and young children — two populations
that are at risk for disease.
• Thiacetazone is associated with life-threatening reactions in patients infected with HIV-1.
Compatibility with antiretrovirals
• Rifampicin alters the metabolism of multiple drugs, particularly protease inhibitors.
Infrequent dosing
• Prospective antibiotics, such as linezolid, might not be useful if they require more than a single dose each day.
Activity against drug-resistant strains
• Drugs such as PAS have been revived owing to the increase in multidrug-resistant and extensively drug-resistant disease.
Activity that is not necessarily in vitro
• Pyrazinamide is a highly effective drug for patients even though it has poor activity under standard laboratory
conditions.
Rapid clearance of chronic infection
• All available drugs, with the exception, possibly, of rifampicin, have limited efficacy in chronic infection. This is
particularly true of agents such as isoniazid that act on the cell wall.
Affordability
• Drugs that are used for other purposes, such as linezolid, are extraordinarily expensive. At current prices, it would be
impossible for them to be used in most areas of the world in which TB is prevalent.

In the case of PA‑824, sequencing the genome of resistant
mutants using a DNA microarray method showed that
mutations in a conserved hypothetical gene produced
resistance11. However, the encoded protein was not the
target of PA‑824, but instead was a nitroreductase that
was responsible for the activation of PA‑824.
Genetic analysis might prove to be an alternative
method for defining new TB drug targets. Using a range
of methods, investigators have found several gene prod-
ucts that, if inhibited, could decrease bacterial growth
or increase host survival after infection12–14. Genes that
are required for growth in vitro are difficult to define
using traditional genetic methods, as mutations in these
genes, by definition, result in clones that are unable to
grow. However, negative screens that identify genes that
cannot be mutated have yielded a set of several hundred
candidate genes that are required for in vitro growth14,15.
Of course, these are simply screens and do not repre-
sent proof that individual gene products are useful as
drug targets. The extension of these screens to in vivo
animal models of TB would be a giant step forwards in
target identification. However, to validate putative tar-
gets in the absence of a known inhibitor it is useful to
construct conditional mutants. Fortunately, recent work
has provided conditional promoters that can be used to
construct these informative strains16–18.
Is it truly important to define single targets for poten-
tial antibiotics? After all, it might be difficult to evolve
resistance to drugs that hit multiple targets (so-called
dirty drugs) and, in fact, there is evidence that some
current drugs, such as INH19,20 and para-aminosalicyclic
acid21, are capable of inhibiting multiple proteins.
However, even in these situations, it is unclear if this
provides a strong advantage as resistance can still arise
from mutations that seem to affect only single putative
targets22,23, perhaps because these represent the points of
greatest vulnerability. In any case, for drugs that hit either
single or multiple targets, it might be difficult to identify
targets using systematic approaches.
High-throughput screening. High-throughput screening
against target proteins has an important role in modern
drug discovery24,25. For M. tuberculosis, biochemical
high-throughput screening has contributed to the find-
ing of two clinical drug candidates, TMC207 (Ref. 10)
and SQ109 (Ref. 26), and has been used to investigate the
viability of small molecules as modulators of a number
of mycobacterial targets.
Two different approaches — the targeting of whole-
cell growth and enzyme inhibition — have been used
individually and together to apply high-throughput
screening to TB drug-discovery efforts. Whole-cell
Box 4 | Gates’ vision
Bill Gates commented at the 2005 World Health Assembly:
“Today, we have tuberculosis drugs you have to take for
9 months. Why can’t we find one that works in 3 days?”

nature reviews | microbiology volume 6 | january 2008 | 43

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REVIEWS

Table 1 | Mechanism of action, resistance and half-life of current first-line antituberculosis agents
Antibiotic Chemical structure Mechanism and target Mutations Half-life Refs
associated with in humans
resistance (hours)
Isoniazid O
H
N Inhibits mycolic acid synthesis; katG (required for 1–3 74
NH2
primary target is InhA and drug activation); inhA
secondary targets are KasA and (promoter mutations);
N
DfrA and others

Rifampicin O HO Inhibits transcription; RNA rpoB 2–3 74

polymerase β-subunit
O
OH O
OH OH
NH
O
H3C N
O N
OH N
O CH3
O

Ethambutol OH Inhibits arabinogalactan embB 3–4 74

H
N synthesis; possibly EmbB
N
H
HO

Pyrazinamide O Unknown (possibly inhibits FAS-I pncA (required for 10 74

N
NH2 or alters membrane energetics) drug activation)
N

Streptomycin H2N NH2 Inhibits protein synthesis; 30S rpsL and rrs 2–3 74
O
N
CHO ribosomal subunit
O
HO OH NHCH3
O OH
OH
H2N N O
OH OH
NH2
OH

Pharmacokinetic profile
A quantitative description of
the fate of a drug from the
moment the treated subject is
dosed with the compound to
the moment when it (and/or its
derivatives) is expelled from
the subject.
screening against either Mycobacterium smegmatis or
M. tuberculosis allows searching for the ultimate goal
— potent growth inhibition or killing. As this type of
screen is not target based, there is a considerable risk
of finding compounds that have generalized toxicity, and
the lack of information on the target will certainly com-
plicate the optimization. This risk might be decreased by
pre-filtering compound libraries27,28 or designing more
targeted screens.
High-throughput screening is being pursued at a
number of facilities, both in industry and academia.
One large National Institutes of Health-funded effort,
the Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (see Further information), offers
a free service to investigators for testing candidate
compounds. The facility has evaluated over 79,000 com-
pounds from more than 9,600 researchers for the inhibi-
tion of M. tuberculosis growth29. Of these compounds,
130 have demonstrated in vitro efficacy against both
drug-sensitive and drug-resistant strains. Such broad
screening efforts hold promise for uncovering new
leads for antituberculars that, from the outset, display
mycobacterial growth inhibition.
High-throughput screening has also been used to
identify inhibitors of a targeted enzyme in a cell-free
environment, through either a binding or functional
assay. High-throughput screening has the potential to
identify hits that could be potent inhibitors of myco-
bacterial growth if the compounds have acceptable
pharmacokinetic profiles and the target is truly essential.
There are many examples of small molecules that have
excellent enzyme inhibition but poor whole-cell potency,
possibly owing to their failure to permeate the myco-
bacterial cell wall. A variant of this strategy relies on
a functional assay in which the inhibition of an entire
pathway can be screened for. For example, a luciferase
reporter can be used to measure the transcription of the
iniBAC operon, which is induced by a diverse set of
mycobacterial cell-wall biosynthesis inhibitors, includ-
ing ethambutol and INH30. Barry and colleagues26 used
this technology to discover SQ109 (FIG. 1), an ethambutol
analogue for which the discovery and clinical progress
will be discussed below.
GlaxoSmithKline and the Global Alliance for TB Drug
Development (TB Alliance; see Further information) have
recently conducted a million-compound screen for new
inhibitors of an M. tuberculosis enoyl-acyl carrier protein
reductase, InhA, which is the target of INH. They found
a high hit rate, probably because the crystallographically
characterized active site can accommodate hydrophobic
groups of varying dimensions, which is consistent with its
acceptance of C16–C56 fatty-acid thioester substrates31,32.
Importantly, these inhibitors should be active against most
of the INH-resistant strains, as they do not require activa-
tion by the catalase–peroxidase enzyme KatG33, which is
required for the activation of INH. Similar screens car-
ried out by GlaxoSmithKline and the Novartis Institute
for Tropical Diseases for inhibitors of isocitrate lyase, an

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© 2008 Nature Publishing Group
 REVIEWS

Table 2 | Mechanism of action, resistance and half-life of selected second-line antituberculosis agents
Antibiotic Chemical structure Mechanism and target Mutations Half-life Refs or
associated with in humans sources
resistance (hours)
Fluoroquinolones O O Inhibits DNA gyrase gyrB Moxifloxacin: 12; 129
F
OH
Gatifloxacin: 8
R N
O
H3C

Ethionamide S NH2 Inhibits mycolic acid ethA (required 2 130

synthesis; InhA for drug
activation) and
inhA (promoter
N
mutations)
Cycloserine H2N
O Inhibits peptidoglycan alr 10 DrugBank
synthesis by blocking (overproduction) (see Further
NH
O
the synthesis and use of and ddl information)
d‑alanine (Ala); Ala racemase (overproduction)
and d-Ala-d-Ala ligase
Para‑aminosalicylic NH2 Inhibits folate metabolism; thyA 0.75–1 131
acid possibly dihydropteroate
synthase
OH

HO O

Capreomycin NH2 Inhibits protein synthesis; tlyA and rrs 4–6 The Merck
R
O methylated nucleotides in Manuals
NH HN O
both ribosomal subunits Medical
R = H, OH
H
N NH2
Library (see
HN O HN Further
H H
H2N
N N
O
N information)
NH2 O O
HN

H 2N O

Kanamycin OH
OH Inhibits protein synthesis rrs 2 132
HO O HO OH
O
H2N HO OH NH2
O O
H2N NH2

Amikacin OH
OH Inhibits protein synthesis rrs 3 133
HO O HO OH
O
H2N HO OH NH2
HO H O O
N NH2

H2N O

Pharmacophore
The chemical functional group
(or groups) that is present on a
molecule and that enables its
biological activity.
Chemotype
A chemical functional group or
classification of a specific array
of functional groups.
enzyme that is crucial for bacterial persistence during
infection34,35, were considerably less successful. Again, this
might be explained structurally, as the active site of this
enzyme is shallow and highly charged. Small, hydrophilic
molecules, such as those that are predicted to bind to
isocitrate lyase, are under-represented in most screening
libraries, as they are less likely to have desirable pharma-
cological properties. Finally, a high-throughput screen for
inhibitors of pantothenate synthetase, an enzyme that is
crucial for the biosynthesis of the essential cofactors acyl
carrier protein and coenzyme A, was recently reported
by White and colleagues36. Of approximately 4,000 com-
pounds assayed, one lead compound was identified and a
preliminary structure characterized in complex with the
target enzyme. This successful outcome suggests a path
forward for the structure-based design of more potent
analogues that inhibit the enzyme and M. tuberculosis.
Combining whole-cell and target-based screens
might avoid the drawbacks of each. For example, the
library that was used to discover SQ109 was screened
using both growth-inhibition and cell-wall-biosynthesis
assays37. Of nearly 5,000 compounds screened, 25 small
molecules were active in both screens, but only one,
SQ775, showed significant activity in a mouse model
of infection.
Structural biology and virtual screening. Describing
the proteome of M. tuberculosis has been the focus
of much research in the past few years. Primarily
owing to the efforts of the TB structural-genomics
consortium, more than 260 X‑ray crystal structures
of interesting proteins, a large percentage of which
were selected on the basis of their being potential
drug targets, have been completed, and many are
currently being used to facilitate medicinal‑chemistry
efforts to rationally design new antibiotics38 (FIG. 1).
The availability of these structures provides the
opportunity to carry out virtual screening. This

nature reviews | microbiology volume 6 | january 2008 | 45

© 2008 Nature Publishing Group
REVIEWS

O O
CH3 O O
N CH F
OH OH F
3
Br OH
H
N N
N N
N O O H
H3C HN O N
CH3 H3C N
NH H H
TMC207 Moxifloxacin Gatifloxacin SQ109

O HO NO2
O HO
N N
O N
OH O N
OH O O OH
OH O SQ775
NH OH OH
O NH Metronidazole
H3C O O
O N
H3C N
N O
O
O OH N N CF3
O HN N
O N
HO N N
N O
Rifalazil Rifapentine Sudoterb

F3C
O2N N O N O
O O2N O
CF3 O
N N O O N N
O H
N N
O F
CH3 O O
PA-824 OPC-67683 O Linezolid
N H3C OH
H3C N O
N N

N S
N Cl CH3 N CF3
H O O NH
O O
OH OH HN
S S O Sulbactam O
N S N N
Chlorpromazine CH3 Trifluoperazine S
O OH
S H HO H H
Thioridazine Clavulanic acid Imipenem

Figure 1 | Chemical structures of non-approved antituberculars.

Nature Reviews | Microbiology

Lipinski’s rules
A set of delimited
physiochemical properties
described by C. A. Lipinski that
best fit a studied subset of
drugs. In general, compounds
that adhere to these guidelines
are said to be drug-like.
Shikimate pathway
A series of biochemical
reactions in plants and
microorganisms that are
involved in the biosynthesis of
aromatic amino acids.
powerful technique has had a beneficial impact on had a minimum inhibitory concentration (MIC) of
numerous drug‑discovery efforts39–41. Screening using 25 µg per ml. Using a different mathematical model,
computational methods can be complementary to a García-García et al. tested 5,000 commercially available
biochemical high-throughput screen because of the compounds and found 18 virtual hits43; 5 of these had an
potential for screening a larger chemical space quickly MIC90 of less than 50 µM.
and inexpensively. Virtual screening can be used in Other groups have instead focused on specific
two ways: to identify compounds that are consist- targets that have known structures. Agrawal and co-
ent with a pharmacophore model irrespectively of the workers45 performed a virtual screen for inhibitors of
identity and structure of the pertinent protein target M. tuberculosis chorismate mutase, which is part
or targets; or to develop inhibitors of a protein based of the essential shikimate pathway in M. tuberculosis46.
on its known three-dimensional structure. They started with known inhibitors of the homologous
Recent examples of virtual screening that were based Saccharomyces cerevisiae enzyme47 and conducted a
on known chemotypes include work from Manetti and three-dimensional pharmacophore search of a database
colleagues42 and García-García and colleagues43. Manetti that has more than 15,000 members. Of the 15 mol-
et al. used a training set of 471 small molecules that had ecules that scored highest, 4 demonstrated inhibition
a range of in vitro activities against M. tuberculosis to in an enzyme assay. Lin and colleagues48 focused on the
construct a model that could be searched using a vir- elucidation of new small-molecule inhibitors of AccD5,
tual library of compounds that was filtered to follow an acyl-CoA carboxylase essential enzyme that cataly-
Lipinski’s rules44. The virtual hits were assayed against ses the transformation of acetyl-CoA and propionyl­
M. tuberculosis, and the two most potent compounds CoA to the corresponding malonyl-thioester and

46 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group

Figure 2 | Overlay of small-molecule inhibitors of InhA. A cross-section through
Nature
the surface of the active site of Mycobacterium tuberculosis Reviews
fatty-acid | Microbiology
enoyl-acyl
carrier protein reductase (InhA), coloured by atom type (carbon, grey; nitrogen, blue;
oxygen, red; and sulphur, yellow). Superimposed are the bound conformations
of NADH50 (carbon, yellow; nitrogen, blue; oxygen, red; and phosphorous, aqua),
C16 fatty acyl substrate analogue trans‑2-hexadecenoyl-(N-acetylcysteamine)
thioester32, shown in its bent conformation (carbon, yellow; sulphur, orange;
nitrogen, blue; and oxygen, red) and 12 inhibitors (carbon, grey; nitrogen, blue;
oxygen, red; sulphur, yellow; and fluorine, purple) that have half-maximal inhibitory
concentration values ranging from 50 nM to 5 µM. Unlike isoniazid and
ethionamide, these are non-covalent reversible inhibitors that form ternary
complexes with enzymes and NAD+. This figure was created using the molecular
modelling system Chimera128.
methylmalonyl-thioester, respectively. They screened over
4 million compounds for binding to either the predicted
biotin or propionyl-CoA binding pockets and found that
1 of the 9 compounds that scored highest had an enzyme
half-maximal inhibitory concentration (IC50) of 10 µM.
Approaches such as these underscore the usefulness
and potential of virtual screening to enable new hits for
drug-discovery efforts.
Each of the discovery technologies discussed in this
section has potential as a starting point for the discov-
ery of a new antitubercular that, on successful passage
through the pre-clinical drug-development pathway, can
produce a clinical candidate. Undoubtedly, the most
efficient and expeditious way to seed drug discovery lies
in the cooperative union of these methodologies in mov-
ing from the validated drug target to a clinical candidate.
Lead optimization This has been highlighted by examining retrospectively a
The process by which a GlaxoSmithKline antibacterial high-throughput screen-
promising small-molecule ing campaign, which, over a wide range of targets, pro-
entity is structurally modified
vided few compounds for follow up49. Additionally, lead
to obtain drug-like
pharmacokinetic, optimization is often a labour- and time-intensive process
pharmacodynamic and safety that many programmes do not endure.
profiles. A detailed understanding of the drug target (or
targets) and how the binding of a drug inhibits target
Efflux pump
An active transport system for
function helps immensely in developing a lead. Whereas
the removal of toxic molecules, efforts to develop new InhA inhibitors, which, in the
such as antibiotics, from cells. future, could complement and/or supplant INH, have
nature reviews | microbiology volume 6 | january 2008 | 47
© 2008 Nature Publishing Group
REVIEWS
yet to yield a clinical candidate, X‑ray crystallography
and structure-based designs have had crucial roles in the
discovery of new inhibitors of this validated TB drug
target32,50–53. Sullivan and co-workers54 have translated
these insights into the discovery of potent triclosan-
based antituberculars. FIG. 2 depicts a select subset of
X‑ray structures that have had a prominent role in this
developing story.
New TB drugs in clinical trials
A chemical entity that has been discovered as an antitu-
bercular by the application of one or more of the methods
discussed above and has cleared the considerable hurdles
in pre-clinical development can enter clinical trials if it has
the approval of the pertinent governmental body (in the
United States, the Food and Drug Administration (FDA)).
The scientific community is hopeful that the drug candi-
dates described below (the structures of which are shown
in FIG. 1) will eventually be used in new treatment regimens
that meet the goals discussed earlier. It is clearly a testa-
ment to the financial commitment of the involved funding
organizations that these candidates are being supported
through a process that is incredibly costly.
Fluoroquinolones. Fluoroquinolones, which have
been used for the treatment of TB since the 1980s55,
currently constitute the second line of defence against
M. tuberculosis and play a key part in the treatment of
MDR disease. Resistance to fluoroquinolones has been
attributed to mutations in the genes that encode gyrase
(gyrA and gyrB)56,57. As quinolone efflux pumps may also
have a role in resistance, it is noteworthy that the annota-
tion of putative pumps in the M. tuberculosis genome58
and the studies of Jacobs and co-workers59 on a probable
efflux pump, lfrA.
Two approved fluoroquinolones, moxifloxacin
and gatifloxacin, have shown promising results both
for treating resistant disease and, possibly, shortening
the course of therapy. Phase II and III clinical trials
that are currently underway are testing the efficacy of
moxifloxacin and gatifloxacin as replacements for either
INH or ethambutol in first-line therapy60,61. These com-
pounds are particularly attractive, as they have already
been approved for use in various other infections and are
known to be safe. As fluoroquinolones are prescribed for
numerous respiratory infections, many cases of TB could
be treated with this monotherapy before they are diag-
nosed. Theoretically, this could lead to drug resistance,
although, as yet, widespread fluoroquinolone-resistance
mutations have not been observed.
Rifampicin analogues. Rifapentine was approved by
the FDA for the treatment of M. tuberculosis infection
in June 1998. The piperazinyl hydrazone functional-
ized rifampicin analogue, initially named DL 473,
was first reported in 1975 (Ref. 62) and subsequently
has been shown to exhibit in vitro antimycobacterial
efficacy that is, in most cases, superior to that of its
parent63–65. A noteworthy advantage that rifapentine
has compared with rifampicin is its longer serum half-
life66, which has encouraged its examination in clinical
REVIEWS
Structure–activity
relationship
(SAR). The relationship
between the chemical structure
of a compound and its
biological or pharmacological
activity. This type of
relationship can be assessed
by considering a series of
molecules, each with a slightly
different structure, and then
noting the effect on the
biological activity that is
associated with each structural
variation.
Fast-track status
The FDA status that is reserved
for products that demonstrate
the potential to treat a serious
or life-threatening condition.
F0 subunit of atp synthase
The transmembrane portion   status and is in Phase I clinical trials.
of the enzyme complex that is
involved in the biosynthesis of
ATP, which has a role in the
passage of protons through   Johnson Pharmaceutical Research and Development10,
the membrane.
Ames mutagenicity test
A sensitive biological method
for measuring the mutagenic
potency of chemical
substances.
48 | january 2008 | volume 6 www.nature.com/reviews/micro
settings to determine its potential for positively altering
the frequency of the antitubercular treatment regimen.
An extension of a Phase III trial that is currently evaluat-
ing rifapentine and INH in the treatment of latent TB
involves recruiting children (C. Dukes Hamilton, per-
sonal communication) to assess the efficacy, pharma-
cokinetic and safety profiles of the drug combination.
Rifalazil. Rifalazil, formerly known as KRM‑1648, has
a benzoxazinorifamycin structure67. This heterocyclic
modification of ansamycin68 is much more potent against
M. tuberculosis clinical isolates than rifampicin69 and is
more efficacious in a mouse model70. Rifalazil presum-
ably has a similar mechanism of action to rifampicin68, as
mutants are cross-resistant69. Rifalazil has a longer half-
life than rifampicin in healthy human volunteers71 and,
unlike rifampicin and rifabutin, is neither metabolized
by, nor an inducer of, rat and dog hepatic cytochrome
P450 enzymes72. These metabolic observations suggest
that rifalazil can be co-administered with drugs that are
sensitive to oxidative metabolism, thereby reducing the
potential for adverse drug–drug interactions. This is a
particularly important consideration, as co-infection
with HIV-1 and TB is common and rifampicin consid-
erably lowers the serum levels of antiretroviral protease
inhibitors73. Rifalazil, however, is not currently registered
with the FDA for a clinical trial of TB, probably owing
to the toxicity that was observed during a Phase II trial
in Brazil74. ActivBiotics is currently investigating signifi-
cantly lower once-weekly doses of rifalazil in Phase II/
III studies to ascertain the effect on patients that have
carotid atherosclerotic disease and have been infected
with Chlamydia pneumoniae (A. Sternlicht, personal
communication). It remains to be seen whether these
lower doses, if safe, would be efficacious for TB.
SQ109. SQ109 was discovered using a high-throughput
screen of ethambutol analogues that had the dual goals
of inhibiting mycobacterial cell-wall synthesis and cell
growth in general26. Barry and colleagues26 used the
structure–activity relationship (SAR) study results reported
by Lederle Laboratories75,76 to prepare more than 63,000
compounds. The compounds were assayed for the inhibi-
tion of M. tuberculosis growth and cell-wall biosynthesis.
They found 119 hits in the cell-wall-biosynthesis assay
and 60 hits in the growth assay that were at least as potent
as ethambutol. SQ109, the optimal hit, shares some struc-
tural similarity with ethambutol, but has superior in vitro
and in vivo activity77. In fact, DNA microarray analysis
suggests that this compound has a different mode of
action from ethambutol9. SQ109 has achieved fast-track
TMC207. Originally named R207910 by Johnson and
TMC207 is a new antitubercular agent from the dia-
rylquinoline (DARQ) class. The drug was found by a
high-throughput screen of a library of approximately
10,000 compounds (K. Andries, personal communi-
cation), during a search for those that inhibited the
growth of the rapidly growing environmental bacterium
© 2008 Nature Publishing Group
M. smegmatis. It is intriguing to note that the first mem-
ber of this DARQ class was isolated as a side product in
chemical experimentation that was designed to prepare
compounds for other early discovery programmes, thus
highlighting the importance of screening (targeted
compounds and side products) across projects.
TMC207 achieves impressive in vitro and in vivo effi-
cacy against drug-sensitive and drug-resistant strains of
M. tuberculosis. Resistance studies identified the biologi-
cal target as the F0 subunit of ATP synthase that is encoded
by atpE10,78,79. This finding, together with the lack of cross-
resistance of TMC207 with existing antitubercular drugs,
highlights the potential that undirected screens have for
the discovery of compounds that have new mechanisms
of action. A Phase II trial is currently underway to
investigate the efficacy of TMC207-containing regimens
versus standard antitubercular therapy in patients who
have MDR TB. Given the previously reported animal
studies80, TMC207 could be added to a second-line treat-
ment regimen or could replace a first-line drug to shorten
the length of treatment.
Sudoterb. The tetra-substituted pyrrole Sudoterb was
discovered by building on the previously described
antimycobacterial SAR of pyrroles81. The N‑substituent
is similar to INH, as both contain an isonicotinoyl
hydrazide moiety. Because the only publicly available
information about this compound comes from the pat-
ent application82, we know little about its mechanism of
action. However, the absence of cross-resistance with
existing therapies and the unique chemical structure sug-
gests that it could be novel. Sudoterb has been reported
to be more potent than INH as a monotherapy in a
mouse model for TB and displays an acceptable phar-
macological profile in mice and dogs83. The compound
is reportedly in Phase I clinical trials83.
Nitroimidazoles. Metronidazole has frequently been
used to treat infections of microaerophilic or anaerobic
bacteria84. Owing to the proposed relevance of limiting
oxygen conditions to the latent phase of TB85, metroni-
dazole has also been investigated as an antitubercular.
Metronidazole kills only dormant M. tuberculosis and
not actively growing cultures86. The drug had no effect
on the growth of M. tuberculosis-infected mouse macro-
phages, but a measurable, although small, efficacy in a
mouse model of chronic-stage M. tuberculosis87. Despite
these mixed results, a Phase II clinical trial in South
Korea is currently recruiting patients to examine the
effect of adding metronidazole to a standard second-line
therapeutic regimen (see ClinicalTrials.gov in Further
information for details of an ongoing clinical trial).
Attempts to discover other nitroimidazole-based
antituberculars led researchers at Ciba-Geigy to discover
CG‑17341 — a nitroimidazooxazole that displays potent
activity both in vitro and in vivo against drug-sensitive
and drug-resistant strains88,89. A toxicity issue noted
in an Ames mutagenicity test hindered this compound
from being developed further. Stover and colleagues90
removed this concern, however, with the discovery of the
potent antitubercular PA‑824 of the nitroimidazopyran

class. In vivo studies demonstrated comparable activity
between PA‑824 and INH91. Most importantly, PA‑824
displays no cross-resistance with existing TB drugs and
is efficacious against non-growing M. tuberculosis under
hypoxic conditions. Intriguingly, this activity against
persistent mycobacteria is not shared with the original
lead compound CG‑17341. However, in vivo tests using
a mouse model have failed to demonstrate an advan-
tage in including PA‑824 with first-line antituberculars
in terms of shortening the duration of treatment 92.
Mechanistically, the mycobacterial target (or targets)
of PA‑824 is unknown. Biochemical evidence suggests
that both protein and lipid synthesis are inhibited by a
compound (or compounds) that is generated from the
bioreduction of the imidazole nitro group84,90,93. Fatty-
acid analysis of treated mycobacteria suggested the inhi-
bition of hydroxymycolate oxidation to ketomycolate90.
Further mechanistic work is required to understand the
mechanism of action of PA‑824. Overall, the likelihood
of a novel mechanism of action and activity against per-
sistent mycobacteria bodes well for the positive impact
of PA‑824 on antitubercular chemotherapy. This com-
pound is in Phase I clinical trials that are supported by
the TB Alliance.
OPC‑67683. Building on the work of Ciba-Geigy in
nitroimidazoles and the discovery of PA‑824, researchers
at the Otsuka Pharmaceutical Company in Japan
examined the SAR around PA‑824 and proposed that
further variation of the furan portion of the hetero-
cycle could yield potent antituberculars that are free
of mutagenicity93,94. Whereas PA‑824 features a pen-
dant 4‑trifluoromethoxybenzyloxy group, the Otsuka
researchers introduced a 4‑piperidine moiety in place
of the trifluoromethoxy group to improve oral bioavail-
ability. OPC‑67683 was eventually prepared in an effort
to decorate the piperidine 4‑position with hydrophobic
groups such as 4‑trifluoromethoxyphenyl.
OPC‑67683 is free of the mutagenicity of its nitroimi-
dazole progenitor, is orally bioavailable in mice and
provides efficacy that is equivalent to rifampicin against
M. tuberculosis Kurono at less than one-fifteenth of
the dose93. OPC‑67683 was equally efficacious against
cultures of drug-sensitive and drug-resistant strains
of M. tuberculosis and superior to INH, ethambutol,
rifampicin, streptomycin, CGI‑17341 and PA‑824, and
also produced rapid eradication of infection in mice.
OPC‑67683 was not metabolized by a panel of human
microsomes and did not positively or negatively interfere
with their catalytic activities, thereby lending hope to the
idea that this drug could be used in combination with
HIV‑1 therapies. Mechanistically, OPC‑67683 seems to
inhibit mycolic-acid biosynthesis and, more specifically,
methoxy- and keto-mycolic-acid biosynthesis93, although
it probably requires biotransformation for its activity95.
Given the limited amount of target information that is
currently available, it is unclear if OPC‑67683 and PA‑824
have different mechanisms of action. OPC‑67683 suc-
cessfully completed Phase I clinical trials, demonstrating
satisfactory safety and pharmacokinetic profiles in healthy
individuals. The small molecule is now being evaluated
nature reviews | microbiology volume 6 | january 2008 | 49
© 2008 Nature Publishing Group
REVIEWS
for its early bactericidal efficacy in Phase II studies in
combination with the standard front-line regimen.
The clinical candidates outlined in this section hold
promise as next-generation antituberculars, but each
must now pass through the phases of clinical trials to
receive approval. This process is not without considerable
uncertainty, as demonstrated by the high attrition rates
for clinical compounds in the United States.
Approved non-TB drugs as antituberculars
A strategy to reduce the risk that is associated with
failure in clinical trials owing to an inadequate human-
safety profile lies in the use of already-approved drugs
as antituberculars. In this section, we will discuss the
potential for three classes of non-TB therapeutics in
the fight against TB. The main hurdle has become the
demonstration of sufficient efficacy at a dosage level that
was previously deemed to be safe.
Linezolid. Linezolid received FDA approval for MDR
Gram-positive bacterial infections in 2000. This
3‑aryl‑2-oxazolidinone antibiotic, and its analogues, has
displayed promising in vitro and in vivo efficacy against
drug-sensitive and drug-resistant M. tuberculosis96,97. In
Staphylococcus aureus, and presumably other bacteria
such as M. tuberculosis, linezolid inhibits protein syn-
thesis by binding to the 23S ribosome to prevent transla-
tion98. Clinical examination of linezolid as a potential
antitubercular, although demonstrating promising
efficacy against MDR and XDR TB, has detected sig-
nificant toxicity99–102. The rate of incidence and severity
of these adverse events might be reduced by decreasing
the dosage amount of linezolid. A clinical trial that is
expected to be completed in late 2007 is currently being
conducted in Brazil to determine the efficacy of lower
drug doses (J. Johnson, personal communication). In
addition, further optimization of the oxazolidinone
series for M. tuberculosis activity is underway103.
The potential for using already approved drugs
as antitubercular agents holds considerable promise.
Given that marketed drugs have well-documented
and acceptable safety profiles, a major hurdle has been
removed in terms of finding new therapies for TB.
However, as most antibiotics have little activity against
M. tuberculosis, the crucial step in using these drugs is
to demonstrate good efficacy.
β-lactam. Although the β‑lactam class of antibiotics
has been used in clinics for over 60 years, none of its
representatives has been used for the treatment of TB.
β‑lactams act by inhibiting bacterial-cell-wall biosynthe-
sis and would be a welcome addition to the antitubercular
arsenal. Unfortunately, M. tuberculosis produces only a
single β‑lactamase that has broad specificity104–106. Other
genes may also have a role in resistance by affecting cell-
wall permeability and/or binding affinity for the perti-
nent penicillin-binding proteins107. Although β‑lactams
can penetrate the cell and inhibit their targets108, their
potency is primarily limited by β‑lactamase-mediated
degradation. Two strategies could avoid this problem.
First, in some cases, β‑lactamase inhibitors, such as
REVIEWS

clavulanic acid and sulbactam, allowed growth inhibi-
tion below the µg per ml level if used in combination
with β‑lactams. Second, the carbapenem imipenem is
resistant to cleavage. Unfortunately, both imipenem108–110
and amoxicillin in combination with clavulanic acid111
have produced mixed results in early bactericidal-
efficacy assessments in humans. However, this remains
a promising area for investigation and recent structural
work using M. tuberculosis and Mycobacterium fortuitum
β‑lactamases106,112 might prompt the further design
of β‑lactamase inhibitors and/or β‑lactams that have
reduced β‑lactamase susceptibility.
Phenothiazines. Phenothiazines have a rich and diverse
history of medicinal uses113 and antitubercular activ-
ity against drug-sensitive and drug-resistant strains
of M. tuberculosis114,115. The major examples are chlo-
rpromazine115, thioridazine116 and trifluoperazine117,118.
These drugs have substantial, and sometimes even
disabling, side effects that limit their use at effective
plasma concentrations. However, they might be more
effective in vivo than in vitro as they are concentrated in
macrophages119,120.
Phenothiazines appear to target the type‑2 nico-
tinamide adenine dinucleotide (NADH):menaqui-
none dehydrogenase (NDH‑2) 121. NDH‑2 is crucial
to the mycobacterial electron-transport chain and is
the only such enzyme in M. tuberculosis that is absent
in humans. Notably, this target seems to be important in
starved cultures122, suggesting that NDH‑2 could be
an attractive target for other compounds. One way to
achieve this would be to start with the known active
phenothiazines and construct analogues that have more
selectivity, a goal that is currently being pursued123,124.
Conclusions
M. tuberculosis would seem to be a vulnerable organism,
given that it has no noteworthy animal or environmental
reservoir and limited genetic diversity. However, despite
the availability of several effective antibiotics, TB con-
tinues to be a widespread and devastating disease. The
need for new fast-acting drugs is clear. Fortunately,
several chemical entities are currently in clinical trials,
and numerous promising compounds are in the earlier
stages of drug development. It is likely that new drugs will
become available in the near future that can cope with
the resistance problem as it currently exists. However,
resistance continually evolves. Persistence is currently
an even more formidable enemy, requiring a much more
thorough understanding of its basic biological underpin-
nings and the small molecules that can modulate its role
in the disease state. Therefore, little hope exists in the
short term for a drastic reduction in the time for treat-
ment, given the targets and drugs that are under clinical
investigation. Despite the considerable challenges that are
posed by these ‘bugs’, the recent improvement in the scale
of efforts to find new drugs lends hope that science will
deliver on the promise of eradicating ‘The Great White
Plague’ that was celebrated prematurely by some owing
to Selman Waksman’s Nobel Prize.

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Acknowledgements
The authors thank M. Spigelman, R. Goldman, J. Garcia,
K. Andries, C. Dukes Hamilton, J. Guilemont, J. Johnson and
A. Sternlicht for insightful conversations and, in some cases,
providing unpublished results. The authors are supported by
a grant from the National Institutes of Health (PO1A1068135)
and the Robert A. Welch Foundation.
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Chlamydia pneumoniae | Mycobacterium smegmatis |
Mycobacterium tuberculosis | Saccharomyces cerevisiae |
Staphylococcus aureus
FURTHER INFORMATION
James C. Sacchettini’s homepage: http://puffer.tamu.edu/
ClinicalTrials.gov: http://clinicaltrials.gov/ct2/show/NCT0
0425113?term=Metronidazole&rank=3
DrugBank: http://redpoll.pharmacy.ualberta.ca/drugbank/
TB Alliance: http://www.tballiance.org
The Merck Manuals Medical Library: http://www.merck.
com/mmpe/index.html
Tuberculosis Antimicrobial Acquisition and Coordinating
Facility: http://www.taacf.org
All links are active in the online pdf
 REVIEWS

Salmonellae interplay with host cells

Andrea Haraga*, Maikke B. Ohlson‡ and Samuel I. Miller*‡§
Abstract | Salmonellae are important causes of enteric diseases in all vertebrates.
Characterization of the molecular mechanisms that underpin the interactions of
salmonellae with their animal hosts has advanced greatly over the past decade, mainly
through the study of Salmonella enterica serovar Typhimurium in tissue culture and animal
models of infection. Knowledge of these bacterial processes and host responses has
painted a dynamic and complex picture of the interaction between salmonellae and
animal cells. This Review focuses on the molecular mechanisms of these host–pathogen
interactions, in terms of their context, significance and future perspectives.

Pinocytosis
A nonspecific process by which
small volumes of extracellular
fluid are taken up by certain
eukaryotic cells owing to the
engulfment of fluid in small
membrane vesicles.
Tight junction
The connection between two
adjacent cells in a monolayer
that is formed by extracellular-
matrix and protein complexes;
impermeable to water and
other molecules.
Macropinocytosis
Used to refer to the
endo­cytosis of large volumes   layer and evade being killed by digestive enzymes, bile
of extracellular fluid and
particles by membrane ruffles.
*Department of Genome
Sciences, ‡Department of
Microbiology, §Department of
Medicine, University of
Washington, Seattle,
Washington 98195, USA.
Correspondence to S.I.M.
e‑mail: millersi@u.washington.
edu
doi:10.1038/nrmicro1788
Published online   tinal inflammatory responses, probably contributes
19 November 2007
Salmonellae are Gram-negative bacterial pathogens
that are capable of infecting a wide range of animals,
which can result in several manifestations of disease1.
The host specificities and the disease symptoms that are
caused by some of the thousands of Salmonella serovars
are listed in TABLE 1. Salmonellae are typically acquired
 
by the oral ingestion of contaminated food or water;
however, exposure to pet reptiles and amphibians, which
are often carriers of the bacteria, can also pose a risk for
salmonellosis in humans (FIG. 1). Although conditions that
 
increase the gastric pH reduce the infectious dose of the
bacterium, salmonellae have an adaptive acid-tolerance
response that might promote their survival in the low
pH milieu of the stomach2. After entering the small
intestine, salmonellae traverse the intestinal mucous
salts, secretory IgA, antimicrobial peptides and other
innate immune defences in order to gain access to the
underlying epithelium3–5. Salmonellae have the ability to
invade the non-phagocytic enterocytes of the intestinal
epithelium by bacterial-mediated endocytosis6. After
adherence to the apical surface of the cell, using vari-
ous fimbrial adhesins, the bacteria disrupt the epithelial
brush border and induce membrane ruffles that engulf
the organisms7,8. However, salmonellae preferentially
enter microfold (M) cells, which are specialized epithelial
cells that sample intestinal antigens through pinocytosis
and transport them to lymphoid cells in the underlying
Peyer’s patches9,10. Alternatively, they can translocate
through the intestinal epithelia after uptake by CD18-
expressing phagocytes11. Moreover, in vitro, salmonellae
are able to disrupt tight junctions, which seal the epithelial
cell layer and restrict the paracellular passage of ions,
water and immune cells12. This, in addition to intes-
to the induction of diarrhoea. The fact that there are
multiple mechanisms for crossing the intestinal barrier
indicates the importance of this strategy to the lifestyle
of salmonellae.
M-cell-mediated uptake also allows salmonellae
to interact with, and possibly enter, intestinal epithe-
lial cells through their basolateral surface. Epithelial
turnover and shedding then, in turn, could return
the bacteria to the lumen of the intestine, simply
requiring that they survive and, perhaps, replicate
intracellularly. Such mechanisms are possibly impor-
tant for intestinal colonization and even for acute
disease by salmonellae, as a great deal of data, from
both animal and cell culture models, indicate that the
specialized ability to invade and survive in epithe-
lial cells is essential for their pathogenesis 13–17. By
contrast, the importance of phagocytosis and traf-
ficking through CD18-positive cells is unknown and
might represent another mechanism, redundant to
M-cell-mediated entrance, for salmonellae to cross
the intestinal barrier without actively interacting with the
epithelial surface. It is interesting to speculate that this
method may be important for typhoid fever, in which so
few organisms can cause disease but induce only mini-
mal intestinal disturbance in the majority of cases. This
could allow Salmonella enterica serovar Typhi (S.  typhi)
to disseminate systemically in a relatively silent fashion,
as opposed to mechanisms that involve bacterial-medi-
ated endocytosis, which are associated with intestinal
inflammation and diarrhoea13,14.
Once the epithelial barrier has been breached,
Salmonella serotypes that are associated with systemic
illness can enter intestinal macrophages by inducing
macropinocytosis , sensing the phagosomal environ-
ment and activating various virulence mechanisms
in order to survive in the microbicidal environment
of the macrophage 18–25 . This promotes bacterial

nature reviews | microbiology volume 6 | january 2008 | 53

© 2008 Nature Publishing Group
REVIEWS
Table 1 | Examples of Salmonella enterica serovars; their hosts and diseases
Salmonella Host Disease and symptoms
enterica serovar specificity
Typhoid
Typhi; Human- Enteric fever: fever; abdominal pain; transient
Paratyphi restricted diarrhoea or constipation; and a salmon-
coloured maculopapular rash on the trunk
Non-typhoid
Typhimurium; Broad-range Gastroenteritis: abdominal pain; vomiting; and
Enteriditis inflammatory diarrhoea
replication and subsequent dissemination throughout
the reticulo­endothelial system (RES). Infection with
non‑typhoidal strains in healthy human adults, however,
is usually limited to the intestine, where the bacteria
induce an early inflammatory response that results in the
infiltration of polymorphonuclear leukocytes (PMNs)
into the intestinal lumen1,26. Production of the potent
PMN chemokine interleukin (IL)‑8, and, perhaps, other
similar molecules, by the infected epithelia is thought
to be required for this process27. The release of cyto-
toxic granules by PMNs, as well as the effects of various
bacterial molecules in stimulating innate immune
inflammatory responses and manipulating host-
cell processes, may then result in the destruction or
turnover of the intestinal mucosa, which contributes
to inflammatory diarrhoea.
Recent progress has been made in our understanding
of the molecular mechanisms by which salmonel-
lae interact with host cells and manipulate various host
responses that result in the induction of intestinal
inflammatory responses and systemic illness. Specifically,
the identification of bacterial and host proteins that are
involved in such processes as the invasion of epithelial
cells, stimulation and repression of signalling cascades,
sensing of the intracellular environment and establishment
of a niche for intracellular replication have contributed to
insights into the complex pathogenesis of this bacterium,
and will be discussed here in detail.
Reticuloendothelial system The two virulence-associated T3SSs
(RES). The meshwork of A specialized apparatus, named the type III secretion
connective tissue that contains
system (T3SS), is essential to salmonellae pathogen-
immune cells, such as
macrophages, and surrounds esis and the colonization of host tissues. The T3SS
tissues that are associated mediates the transfer of bacterial virulence proteins,
with the immune system, such known as effectors, from the bacterial cell into the
as the spleen and lymph host-cell cytoplasm28. Once inside the eukaryotic cell,
nodes. Immune cells in the RES
provide surveillance of antigens
these effectors can alter host cellular functions, such as
that the body encounters and cytoskeletal architecture, membrane trafficking, signal trans-
can be quickly recruited to duction and cytokine gene expression, in order to promote
sites of infection. bacterial survival and colonization. The T3SS is
evolutionarily related to the flagellar export system and
Pathogenicity island
A large region of genomic DNA is present in multiple Gram-negative animal and plant
that encodes genes that are pathogens. It comprises more than 20 proteins, some of
associated with virulence. A which form a supramolecular structure that is known as
pathogenicity island is typically the needle complex (NC) (FIG. 2). Electron micrographs of
transferred horizontally
between bacterial strains and
purified T3SSs revealed that the NC consists of a multi-
is often inserted into tRNA ring base that spans the inner and outer membranes of
genes within the genome. the bacterial envelope, an inner rod that joins the rings
54 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
and a hollow needle29,30. Another set of proteins comprise
the translocon, which is thought to be involved in the
translocation of effectors by forming a translocation pore
in the host cellular membrane31. The effector proteins
have been shown to contain specific targeting signals
located in their amino termini that route them to the
T3SS32–35. In addition to the short secretion signal, many
effectors have a binding site for a specific chaperone,
the function of which is thought to be to stabilize and
target its cognate substrate to the translocation appara-
tus34,36,37. Most chaperones are specific for a single effec-
tor, but some can facilitate the secretion of more than one
effector protein38–41. An ATPase that is located in the base
of the NC not only drives the export process but also
facilitates the release of effectors from chaperones before
transport42. Although much of the T3SS is similar among
Gram-negative bacteria, the set of effector proteins is
unique to each species. For a complete list of Salmonella
spp. effectors, their host-cell functions and binding
partners, see Table 2.
Salmonellae encode two distinct virulence­­‑associated
T3SSs within Salmonella pathogenicity islands 1 and 2
(SPI1 and SPI2) that function at different times during
infection28. Whereas the SPI1-encoded T3SS is active
on contact with the host cell and translocates bacterial
proteins across the plasma membrane, the SPI2 T3SS is
expressed within the phagosome and translocates effectors
across the vacuolar membrane. The SPI1 system has been
shown to be required for invasion of non-phagocytic
cells, induction of intestinal inflammatory responses
and diarrhoea, as well as colonization of the intestine.
The SPI2 T3SS, by contrast, has an important role in
bacterial survival in macrophages and establishment of
systemic disease. Interestingly, Brown and colleagues43
have recently shown by RIVET (recombination-based
in vivo expression technology) that expression of the
SPI2 T3SS in mice might begin in the early stages of
Salmonella enterica serovar Typhimurium (S. typhimu-
rium) infection, before intestinal penetration. As there
is currently no evidence that this T3SS is also involved
in intestinal colonization, the authors speculated that its
early induction in the intestinal lumen might prepare
the bacterium for the inhospitable intracellular environ-
ment of the macrophage and ease the transition to the
later systemic phase of the disease. However, it is also
possible that early expression of this T3SS is required for
the optimal function of the invasion-associated T3SS, as
mutations in the SPI2 apparatus can cause a significant
reduction in the expression of several SPI1 T3SS genes
and impair the ability of the bacterium to invade epi-
thelial cells44,45. Conversely, there is mounting evidence
that some of the effectors of the SPI1 T3SS are expressed,
or persist within, host cells long after bacterial inter-
nalization and contribute to events that were previously
attributed exclusively to effectors of the SPI2 T3SS46–51.
Although it is unknown whether mutations in the SPI1
T3SS could cause pleiotropic effects, these findings indi-
cate that the two Salmonella T3SSs do not operate in an
independent manner, as previously thought, and pos-
sibly cooperate to facilitate the intracellular lifestyle of
the bacteria. This is particularly intriguing, because the

Salmonella spp.
M cell
Epithelial cell
Macrophage
T cell B cell
Gastroenteritis:
PMN influx
Enteric fever: dissemination
to lymph nodes, liver and spleen
Figure 1 | Biology of salmonellae infection. Orally ingested salmonellae
Nature Reviews |survive at the
Microbiology
low pH of the stomach and evade the multiple defences of the small intestine in order to
gain access to the epithelium. Salmonellae preferentially enter M cells, which transport
them to the lymphoid cells (T and B) in the underlying Peyer’s patches. Once across the
epithelium, Salmonella serotypes that are associated with systemic illness enter intestinal
macrophages and disseminate throughout the reticuloendothelial system. By contrast,
non-typhoidal Salmonella strains induce an early local inflammatory response, which
results in the infiltration of PMNs (polymorphonuclear leukocytes) into the intestinal
lumen and diarrhoea.
sequence information and genetic organization of the
two Salmonella T3SSs, as well as their phylogenetic
distribution among Salmonella species, indicate that
they were acquired independently, at different periods
and from different sources44,52–57. Therefore, it is likely
that selection pressure from animal environments led
to the cooperation between the two T3SSs, to opti-
mize colonization, invasion and intracellular survival
of salmonellae in animals.
The most extensively used animal model for investi-
gating the contributions of the two T3SSs to salmonellae
virulence is the highly sensitive natural-resistance-
associated macrophage protein 1 (Nramp1)‑null mouse,
which is susceptible to mortal infection if inoculated with
as few as ten bacteria58. Nramp1 is a macrophage-specific
ion exporter that removes ions from the Salmonella-
containing vacuole (SCV) and, thus, restricts bacterial
replication59. The Nramp1-null mouse model system
has been used successfully to identify many important
genes that are required for T3SS-associated virulence60,61.
However, it is too sensitive to detect phenotypes for
T3SS effectors that contribute to long-term persist-
ence, because these mice do not survive long enough
to study the proteins that are necessary for systemic
colonization. The resurgence of interest in the resistant
Nramp1-positive mouse model has enabled studies to
be performed that are more physiologically relevant
to long-term systemic infection49,62. These reports
confirmed that SPI2 T3SS effectors are important for
persistent infection and colonization, but also validated
nature reviews | microbiology volume 6 | january 2008 | 55
© 2008 Nature Publishing Group
REVIEWS
older data that had suggested a role for the SPI1 T3SS
during systemic disease17,63. This, combined with the
previously mentioned putative function of the SPI2 T3SS
prior to intestinal penetration, further demonstrates that
the dogmatic compartmentalization of the roles of the two
T3SSs is simplistic. In addition, the recent resurrection of
the streptomycin-treated mouse model of acute intestinal
infection with inflammation and diarrhoea might enable
dissection of the roles of the host and bacteria in acute
intestinal disease64. It is likely that, in the near future,
models of salmonellae-induced chronic intestinal disease
at the intestinal mucosal surface will be further devel-
oped and studied. In fact, our group has demonstrated
that the A/J mouse strain develops this type of disease
(L. Peiser, K. Smith and S.I.M., unpublished observa-
tions). These animal models should be useful in under-
standing the contributions of mucosal immune defences,
as well as bacterial factors such as T3SS effectors, to
chronic intestinal disease. However, these experimental
systems also have limitations, as specific disease outcomes
may be different in different hosts for broad host-range
salmonellae. A future challenge in understanding these
animal models will be to determine the effects of specific
effectors in both the model and natural hosts. In addi-
tion, the diversity and evolution of the effector content of
different Salmonella strains isolated from natural sources
might also reveal different functions or roles that cannot
be explored by these model systems. In this regard, many
S. typhimurium effectors are present in only a percentage
of strains, and in some serotypes, such as Typhi, are only
present as pseudogenes. Selection for the presence or
loss of effectors in specific animal populations or human
disease settings might, ultimately, be more informative of
the actual natural disease or colonization than the phe-
notypes of deletion mutants in model systems. Therefore,
the acceptance of the limitations of these animal mod-
els is important to prevent dogmatic viewpoints about
the roles of such mechanisms or specific T3SS effector
proteins. This is particularly relevant to the study of effec-
tor proteins with redundant functions, as the necessity
for their similar tasks during pathogenesis might not be
apparent in model systems. A complete picture will prob-
ably require the integration of animal model information
with real world information about effector content and
disease course in nature.
Bacterial-mediated endocytosis
The concerted function of at least five SPI1 T3SS effec-
tors is required for efficient invasion of cultured epithe-
lial cells, although optimal invasion in animal tissues
might be more complex and diverse (FIG. 3). SopE, SopE2
and SopB (also known as SigD) activate the host Rho
GTPases Cdc42, Rac1 and RhoG, which leads to actin
cytoskeletal reorganization, membrane ruffling and bac-
terial internalization by macropinocytosis65–70. However,
recent evidence suggests that only Rac1 and RhoG are
indispensable for the actin remodelling events that are
generated by Salmonella spp. during host-cell entry70.
SopE, SopE2 and SopB are all essential for the invasion of
epithelial cells, as an S. typhimurium mutant that is
defective in all three effectors cannot induce actin
REVIEWS
a modulating actin dynamics directly. SipA (also
Needle
Base
b
Base
Needle
Figure 2 | Structure of the Salmonella type III
Nature Reviews | Microbiology
secretion system (T3SS). a | Electron micrographs of a
purified Salmonella enterica serovar Typhimurium
(S. typhimurium) needle complex (NC). b | Electron
cryomicroscopy and surface images of the structures of
the base and NC of the S. typhimurium T3SS. Part a
reproduced, with permission, from Ref. 31 (2002)
Elsevier Science. Part b reproduced, with permission,
from Ref. 185  (2004) American Association for the
Advancement of Science.
rearrangements and, therefore, become intracellular69.
Whereas SopE and SopE2 are potent guanine nucleotide
exchange factors (GEFs) for all three GTPases, SopB
only stimulates Cdc42 and RhoG indirectly through its
phosphoinositide phosphatase activity65,67–70. Although
the mechanism of action of SopB is not yet understood,
Patel and colleagues70 have recently shown that activa-
tion of RhoG by SopB occurs by a cellular exchange
factor called SGEF. As SGEF has a phosphoinositide-
binding pleckstrin homology domain, it is plausible
that it is activated by the phosphoinositide fluxes that are
generated by the enzymatic action of SopB71. Activation
of Rho GTPases then results in the activation of the
Wiskott–Aldrich Syndrome protein (WASP) family
members N‑WASP and WAVE2, which leads to recruit-
ment of the actin-related protein‑2/3 (Arp2/3) complex
to sites of membrane ruffles and stimulation of actin
polymerization67,72–74.
There are two SPI1 T3SS effectors that promote
bacterial internalization by binding to actin and
56 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
called SspA) helps to initiate actin polymerization
at the site of S. typhimurium entry by decreasing the
critical concentration and increasing the stability
of actin filaments, whereas SipC (also called SspC),
an effector that also functions as a translocon and is
inserted in the host cell’s plasma membrane with a
cytoplasmic domain that may bind to intermediate
filaments, can nucleate and bundle actin 75–77. In
addition, SipA can enhance the activity of SipC
independently of any host cellular protein, which
indicates that there might be a unique collaboration
between the two effectors78. Although SipA and SipC
might act in concert with SopE, SopE2 and SopB to
mediate the cytoskeletal changes that are required
for the formation of membrane ruffles, they can-
not induce membrane ruffling and invasion by
themselves 69,76,77,79. Instead, they seem to facilitate
efficient bacterial uptake by directing the spatial
localization of actin foci beneath the invading bac-
teria and, perhaps, preventing disassembly of the
S. typhimurium-induced actin structures.
Intestinal inflammatory responses
The stimulation of Cdc42 by SopE, SopE2 and SopB
also triggers several mitogen-activated protein kinase
(MAPK) pathways, including the Erk, Jnk and p38 path-
ways, which results in the activation of the transcription
factors activator protein‑1 (AP‑1) and nuclear factor-κB
(NF-κB)16,70,80 (FIG. 3). These transcription factors then
direct the production of pro-inflammatory cytokines,
such as IL‑8, which stimulate PMN transmigration and
the inflammatory response leading to diarrhoea. Boyle
and colleagues81 have recently reported that disrup-
tion of tight junction structure and function — one
aspect of S. typhimurium-induced enteritis that is
likely to be important — by the effectors SopB, SopE,
SopE2 and SipA also occurs by activation of Rho fam-
ily GTPases. In addition, SopB also promotes intestinal
disease by increasing the intracellular concentration of
d‑myo-inositol 1,4,5,6-tetrakisphosphate, a compound
that stimulates cellular chloride secretion and fluid
flux69,82. SipB (also called SspB), an effector protein that
is also part of the SPI1 T3SS translocation machinery,
adds to the inflammatory response by increasing pro-
duction of the pro-inflammatory cytokines IL‑1β and
IL‑18 through binding and activating caspase‑1 (Ref. 83).
Caspase‑1 activation can also occur by the recognition of
flagellin by the Ipaf cytoplasmic signalling pathway84,85.
It is plausible that this is the result of the accidental
translocation of flagellin into the host cell’s cytoplasm
by the SPI1 T3SS owing to the conserved evolution of
the flagellar and the SPI1-encoded secretion systems.
The observation that caspase‑1-deficient mice are more
susceptible to orally administered S. typhimurium than
wild-type mice indicates that SPI1 T3SS-mediated cas-
pase‑1 activation could be essential to diarrhoeal disease
and intestinal survival86,87. SopA and SopD also contrib-
ute to enteritis in calves13,88,89. Recent evidence indicates
that SopA has a HECT (homologous to E6-AP carboxyl
terminus)-like E3 ubiquitin ligase activity, which is
 REVIEWS

Table 2 | Effectors of the Salmonella SPI1- and SPI2-encoded type III secretion systems (T3SSs)
Effector Cellular function Host-cell target References
SPI1 T3SS
AvrA Inhibits nuclear factor (NF)‑κB signalling and interleukin (IL)‑8 Unknown 99,186
production; also prevents ubiquitination of β‑catenin
SipA or SspA Decreases the critical concentration of G‑actin and increases the F-actin; T‑plastin 76, 81,187,188
stability of F‑actin; also induces PMN transepithelial migration
and disrupts tight junctions
SipB or SspB* Binds and activates caspase‑1 and induces autophagy in Caspase‑1; cholesterol 83,189,190
macrophages
SipC or SspC* Nucleates and bundles actin F-actin; cytokeratin‑8 and 75,77,191
cytokeratin-18
SopA Stimulates PMN transmigration by HECT-like E3 ubiquitin ligase Unknown 89,90
activity
SopB or SigD Activates Cdc42, RhoG, AktA and chloride secretion through its Unknown 69,70,81,82,192
inositol phosphatase activity and disrupts tight junctions
SopD Stimulates fluid accumulation in bovine ligated ileal loops and Unknown 13,82,91
contributes to diarrhoea in calves and systemic disease in mice
SopE Activates Cdc42, Rac1 and RhoG by its GEF activity and disrupts Cdc42, Rac1 and Rab5 65,81,193
tight junctions
SopE2 Activates Cdc42, Rac1 and RhoG by its GEF activity and disrupts Cdc42 and Rac1 67,68,81
tight junctions
SptP Inhibits Cdc42 and Rac1 by its GAP activity and MAPK signalling Rac1 92,94,95
and IL‑8 secretion through its tyrosine phosphatase activity
SPI2 T3SS
GogB Unknown Unknown 194
PipB Unknown Unknown 157
PipB2 Contributes to Sif formation Kinesin‑1 157,165,170
SifA Induces Sif formation, maintains integrity of the SCV and SKIP and Rab7 154,164,166, 181,195
downregulates kinesin recruitment to the SCV
SifB Unknown Unknown 156
SopD2 Contributes to Sif formation Unknown 91
SpiC* Interferes with endosomal trafficking Hook3 150,151–153
SpvB‡ Actin-specific ADP-ribosyltransferase and downregulates Sif Actin 160, 175,176,196
formation
SseF Contributes to Sif formation and microtubule bundling Unknown 162,169
SseG Contributes to Sif formation and microtubule bundling Unknown 162,169
SseI or SrfH Contributes to host-cell dissemination Filamin and TRIP6 160,197
SseJ Maintains integrity of the SCV and has deacylase activity Unknown 155,180
SseK1 Unknown Unknown 198
SseK2 Unknown Unknown 198
SseL Deubiquitinase Ubiquitin 199
SspH2 Inhibits the rate of actin polymerization and contributes to Filamin and profilin 96,160
virulence in calves
SteA Unknown Unknown 200
SteB Unknown Unknown 200
SteC Unknown Unknown 200
SPI1 and SPI2 T3SS
SlrP Contributes to virulence in calves Unknown 201
SspH1 Inhibits NF‑κB signalling and IL‑8 secretion, contributes to PKN1 95–98
virulence in calves and has E3 ubiquitin ligase activity
*Also a component of the secretion apparatus. ‡Has not been definitively shown to be an SPI2 T3SS effector. GAP, GTPase-activating protein; GEF, guanine
nucleotide exchange factor; HECT, homologous to E6-AP carboxyl terminus; MAPK, mitogen-activated protein kinase; PMN, polymorphonuclear leukocyte; SCV,
Salmonella-containing vacuole; Sif, Salmonella-induced filament; SPI, Salmonella pathogenicity island.

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© 2008 Nature Publishing Group
REVIEWS

a multifunctional protein that is also involved in the

Membrane reversal of the pro-inflammatory signalling cascade
Tight
ruffle
junction that is associated with invasion94. It has tyrosine phos-
Salmonella phatase activity, which has a role in downregulation
spp.
of the S. typhimurium-induced activation of the MAPK
Actin
Erk. In addition, S. typhimurium can downregulate IL‑8
production after invasion of intestinal epithelial cells,
SopE SipA SopA and SptP and SspH1 participate in this process95. SspH1
Epithelial
cell
SopE2 SipC SptP is a leucine-rich-repeat protein that is localized to the
SopB
SopB Rho GTPases mammalian nucleus and inhibits NF‑κB-dependent
gene expression95,96. It has also been shown to have
Rho GTPases MAPK CI– MAPK SspH1
AvrA ubiquitin ligase activity97. As it interacts with a host
NF-κB
CI– NF-κB serine/threonine protein kinase called PKN1, which if
AP-1 activated also inhibits NF‑κB-dependent gene expres-
sion, SspH1 could interfere with inflammatory signal-
ling by binding to and activating PKN1 (Ref. 98). In fact,
IL-1β
IL-8
IL-18 PKN1 is activated in epithelial cells during infection
with S. typhimurium. However, as this also occurs in
Macrophage the absence of SspH1, it is possible that SspH1 is not
SipB required for this process or that there are additional
PMN
effectors or bacterial mechanisms that are capable of
activating PKN1. Another SPI1 T3SS effector, AvrA,
Figure 3 | SPI1 T3SS-induced changes in host cells. On contact with the epithelial has been reported to inhibit NF‑κB activity and pro-
Nature Reviews | Microbiology
cell, salmonellae assemble the Salmonella pathogenicity island 1(SPI1)-encoded type III inflammatory cytokine secretion99. However, unlike
secretion system (T3SS) and translocate effectors (yellow spheres) into the eukaryotic its Yersinia spp. homologue, YopJ, which binds and
cytoplasm. Effectors, such as SopE, SopE2 and SopB, then activate host Rho GTPases, acetylates MAPK kinases and the inhibitor of NF-κB
which results in the rearrangement of the actin cytoskeleton into membrane ruffles, kinase (IKK), thereby preventing their phosphorylation
induction of mitogen-activated protein kinase (MAPK) pathways and destabilization of
and activation, AvrA has not been shown to interact
tight junctions. Changes in the actin cytoskeleton, which are further modulated by the
actin-binding proteins SipA and SipC, lead to bacterial uptake. MAPK signalling with or affect the activity of any host proteins that are
activates the transcription factors activator protein‑1 (AP‑1) and nuclear factor-κB involved in the NF‑κB pathway100,101.
(NF‑κB), which turn on production of the pro-inflammatory polymorphonuclear The fact that salmonellae use multiple effectors to
leukocyte (PMN) chemokine interleukin (IL)‑8. SipB induces caspase‑1 activation in dampen the host’s inflammatory response indicates
macrophages, with the release of IL‑1β and IL‑18, so augmenting the inflammatory that this function is important for their pathogenesis,
response. In addition, SopB stimulates Cl– secretion by its inositol phosphatase activity. perhaps by contributing to colonization of the intestinal
The destabilization of tight junctions allows the transmigration of PMNs from the tract over prolonged periods of time. Furthermore, it is
basolateral to the apical surface, paracellular fluid leakage and access of bacteria to the interesting to speculate that the evolutionary pressure
basolateral surface. However, the transmigration of PMNs also occurs in the absence of on the host–pathogen interaction for non-typhoidal
tight-junction disruption and is further promoted by SopA. The actin cytoskeleton is
salmonellae is for asymptomatic intestinal colonization,
restored and MAPK signalling is turned off by the enzymatic activities of SptP. This also
results in the down-modulation of inflammatory responses, to which SspH1 and AvrA whereas for typhoidal strains, which have poor intesti-
also contribute by inhibiting activation of NF‑κB. Figure reproduced from an original nal colonization, the selective pressure is for replication
kindly provided by A. Haraga, University of Washington, USA. within professional phagocytes, a condition that is also
less inflammatory than extracellular growth. The bacte-
ria might induce a mild state of inflammatory response
involved in the S. typhimurium-induced transepithelial and their goal might be to evolve towards parasitism or
migration of PMNs90. However, how SopD, an effector that commensalism. In fact, the long period of intestinal colo-
is also expressed under SPI2 T3SS-inducing conditions nization greatly exceeds that of the acute disease phase for
and continues to contribute to later stages of infection, non-typhoidal strains102. Also, typhoidal disease in the
exerts its effects remains unknown33,91. In summary, all absence of antibiotic therapy is often characterized by
of these mechanisms strongly implicate the SPI1 T3SS in periods of few or no symptoms, followed by a relapse that
the induction of gastroenteritis and intestinal disease. is more of a chronic, prolonged febrile illness, in which
intracellular replication is the predominant bacterial
Reversal of cytoskeletal and signalling responses lifestyle1. Thus, it is likely that a dynamic induction of
Interestingly, shortly after bacterial invasion the actin inflammatory responses, followed by their dampening, is
Transepithelial migration
cytoskeleton regains its normal architecture7. SptP has important to the diseases caused by salmonellae.
The movement of cells, such as
neutrophils and invading been shown to participate in this reversal process by
bacteria, from the basolateral its GTPase-activating protein activity on Cdc42 and The SCV as a niche for replication
(bottom) to the apical (top) Rac1 (Ref. 92) (FIG. 3). The opposing activities of SopE Salmonellae are capable of infecting and replicating in
surface, or the reverse, of an or SopE2 and SptP are mediated by the temporal regula- many cell types, but are thought to primarily replicate
epithelial cell layer. Migration
can also occur between two
tion of these proteins93. SopE has a shorter half-life in in macrophages, as they are found in the lymphatic tis-
adjacent cells through tight eukaryotic cells than SptP owing to its ubiquitination sues and organs of the RES during systemic infection1,103.
junctions. and rapid proteasome-dependent degradation. SptP is Salmonella strains that are defective for macrophage

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© 2008 Nature Publishing Group
 REVIEWS
Salmonella spp.

intracellularly from hours to days, making it a unique

SspH2 SifA
SpvB PipB2 s macrophages in which the lysosomal compartments
Spacious Ssel tor
phagosome le mo
u
tub
M icro Sif
SCV
Actin SseJ
Epithelial cell
Microtubules
Nucleus
SseF
SseG
Golgi Secretory vesicles
Figure 4 | Formation of the SCV and induction of the SPI2 Nature
T3SSReviews Microbiology
within| the host cell.
Shortly after internalization by macropinocytosis, salmonellae are enclosed in a
spacious phagosome that is formed by membrane ruffles. Later, the phagosome fuses
with lysosomes, acidifies and shrinks to become adherent around the bacterium. This is
called the Salmonella-containing vacuole (SCV), which contains the endocytic marker
lysosomal associated membrane protein 1 (LAMP‑1; purple). The Salmonella
pathogenicity island 2 (SPI2) T3SS (type III secretion system) is induced within the SCV
and translocates effector proteins (yellow spheres) across the phagosomal membrane
several hours after phagocytosis. The SPI2 T3SS effectors SifA and PipB2 contribute to
Salmonella-induced filament (Sif) formation along microtubules (green) and regulate
microtubule-motor (yellow star shape) accumulation on the Sif and the SCV. SseJ is a
deacylase that is active on the phagosome membrane. SseF and SseG cause
microtubule bundling adjacent to the SCV and direct Golgi-derived vesicle traffic
toward the SCV. Actin accumulates around the SCV in a SPI2 T3SS-dependent manner,
in which SspH2, SpvB and SseI are thought to have a role.
replication are avirulent in mouse models of infection,
which underscores the importance of bacterial survival
and replication in macrophages to disease104. In addi-
tion, the observation that many attenuated strains of
salmonellae are auxotrophic, particularly for purines,
pyrimidines, amino acids and other metabolites that
are required for their replication but that are not readily
available within mammalian cells, supports the concept
that intracellular replication is essential to salmonellae
virulence105. Unlike non-phagocytic cells, salmonellae
can enter macrophages by several endocytic processes,
including SPI1 and non-SPI1 T3SS-induced macropino-
cytosis20. Following SPI1 T3SS-induced macropinocyto-
sis, a few SPI1 T3SS effectors, such as SipA, SopB, SopD
Auxotrophic
An organism that cannot
and SopE2, persist within the cell and have recently been
synthesize certain organic implicated in contributing to the intracellular stages of
compounds, such as amino the infection process47,48,50,51,91,106. Once intracellular,
acids, that are necessary for its salmonellae remain inside a vacuolar compartment,
metabolism. For growth,
which has been named the spacious phagosome (SP)20
auxotrophic organisms must
be able to take up the lacking (FIG. 4). The SP shrinks over minutes to hours to form
compound from the an adherent membrane around one or more bacteria,
surrounding environment. which is then referred to as the SCV. The SCV can persist
phagosome with respect to the normal progression of
phagolysosomal maturation and recycling. Although
there has been controversy within the field, several
reports have shown that salmonellae can survive within
have fused with the SCV107–110. Consequently, the avoid-
ance of phagolysosomal fusion is unlikely to be a major
pathogenic strategy of salmonellae. Studies in various
cell types have also demonstrated that the vacuole acidi-
fies; however, depending on the mechanism of host‑cell
entry, vacuolar acidification may be delayed in both
macrophages and epithelial cells19,110–112. The SCV has
been shown to interact transiently with the early endo-
cytic pathway and quickly gain and lose early endocytic
markers, such as EEA1 (early endosomal antigen 1), TfR
(transferrin receptor) and the early endocytic traffick-
ing GTPases Rab5 and Rab11 (Refs 113,114). Several late
endosomal markers are commonly associated with the
SCV at later time points, including the GTPase Rab7,
LAMP1 (lysosomal associated membrane protein 1),
LAMP2, LAMP3 and the vacuolar ATPase110,113,115,116.
There is conflicting data concerning the occurrence of
M6PR (mannose‑6-phosphate receptor), LBPA (lysobi-
sphosphatidic acid) and the lysosomal hydrolase cathep­
sin D on the SCV115,117–120. Furthermore, cholesterol has
been reported to accumulate on the SCV118,121. The pres-
ence or absence of different markers on the persistent
SCV may simply indicate that they are variably detected
rather than reflect whether or not the SCV has matured
through a normal endocytic pathway. As it has been
shown that the SCV can fuse with lysosomes and acidify,
the ability of salmonellae to survive exposure to lyso-
somal contents reveals that resistance to antimicrobial
peptides, nitric oxide and oxidative killing are important
to its survival within macrophages and to virulence122–129.
This is supported by the observations that Salmonella spp.
mutants that are sensitive to these chemicals are attenu-
ated for mouse virulence, whereas mice that are deficient
for the production of these compounds have increased
susceptibility to Salmonella spp.23,104,129–131
Sensing and response to the vacuole
Salmonellae sense the acidic environment of the SCV,
resulting in the induction of various regulatory systems
that promote intracellular survival, for example, by
surface remodelling of the protein, carbohydrate and
membrane components of the bacterial envelope19. Such
regulatory systems include OmpR/EnvZ, PhoP/PhoQ,
RpoS/RpoE, PmrA/PmrB, Cya/Cyp and cyclic diGMP,
all of which confer resistance to antimicrobial peptides
and oxidative stress18,124,127,129,132–135. The phagosomal
environment is acidic, with a pH range of <5 to 5.5, has
a concentration of magnesium and calcium in the 1 mM
range and contains antimicrobial peptides and oxygen
and nitrogen radicals that can damage the bacterial
cell19,111,112. Various studies indicate that both pH and
antimicrobial peptides are important signatures of the
phagosomal environment and such conditions activate
many of the regulators that are implicated in salmonellae
virulence24,25,125,136. It is likely that several sensory systems

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© 2008 Nature Publishing Group
REVIEWS
respond to the phagosome environment and cooperate
to orchestrate the complex cascade of events that are nec-
essary to alter the bacterial surface and promote intracel-
lular survival137. The best characterized of these is the
PhoQ sensor, which promotes resistance to antimicro-
bial peptides18,25. PhoQ contains a novel acidic domain
that is bridged to the inner membrane by interactions
with metal ions and binds to and initiates responses to
antimicrobial peptides138,139. It also responds to pH by
structural changes that are determined by regions of the
protein separate from the metal ion bridges involved in
antimicrobial peptide sensing136.
After phagocytosis, salmonellae undergo extensive
bacterial surface remodelling, as has been shown for the
lipid A component of lipopolysaccharide (LPS) during
growth within macrophages137,140. Bacterial molecules
that the host can recognize as indicators of infection,
such as the SPI1 T3SS and flagellin, are repressed and
the LPS structure is altered85,141. Some of the specific sur-
face modifications include: decreasing the length of the
O antigen, which is the repeating carbohydrate polymer
of LPS; alterations to the number of acyl chains in the
structure of the lipid A component of LPS; and changes
in the protein content of the outer membrane, the inner
membrane and the peptidoglycan layer142–145. Synthesis
of enzymes that allow the bacteria to cope with oxida-
tive and nitrogenous radicals also occurs146. Microarray
studies have shown that up to 919 S. typhimurium
genes are differentially regulated in response to the
phagosomal environment, demonstrating that dra-
matic transcriptional and post-translational changes
probably occur when salmonellae make the transition
from a nutrient-rich extracellular environment to the
intracellular environment147.
Protein delivery across the vacuolar membrane
As a result of sensing the phagosomal environment,
the expression and assembly of the SPI2-encoded T3SS
is induced134,148. Although the function of this T3SS in
pathogenesis remains poorly defined, it has been shown
to be essential for virulence in the mouse model of infec-
tion22,149. As mutants of the SPI2 T3SS cannot replicate
in tissue-culture cells and animal models, it is likely
that the role of this T3SS during disease is to promote
intracell­ular replication within the intestine during the
acute phase of the infection and in other organs during
the chronic state. A reasonable hypothesis for the main
function of the SPI2 T3SS is that it promotes intracellular
replication by altering host vesicular trafficking, so that
useful metabolic molecules, such as amino acids and lip-
ids, are routed to the SCV and the vesicular compartment
membrane is expanded. To date, at least 20 Salmonella
spp. effector proteins are known to be translocated by
the SPI2 T3SS across the phagosomal membrane into the
eukaryotic-cell cytoplasm; however, their specific roles
in promoting intracellular replication or modifying
vesicular movement are not yet understood (for a list
of SPI2 T3SS effectors, their functions and binding part-
ners, see TABLE 2). In addition, no individual translocated
effector has definitively been shown to alter vesicular
trafficking. Although it has been reported that SpiC alters
60 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
endosome–endosome fusion in vitro and binds to pro-
teins that are implicated in vesicular trafficking, its role
as a T3SS effector protein remains controversial150,151. As
it has not been universally observed to be translocated
into eukaryotic cells and it is required for the transloca-
tion of several, if not all, SPI2 T3SS effectors, as well as
the surface expression of translocon proteins, it is more
likely that SpiC is part of the SPI2 secretion apparatus
and is not an effector152,153. The most important trans-
located effectors, by virtue of causing virulence defects
in mice when mutated, are SifA, SseJ, SseF, SseG, SopD2
and PipB2 (Refs 91,154–158) . The observation that
S. typhimurium that lacks any single SPI2 T3SS effector
protein cannot cause the same virulence attenuation in
mice as a mutant strain that lacks the entire SPI2 T3SS
suggests that many effectors function cooperatively to
exert their effects on the host cell. Furthermore, the
deletion or mutation of many effector genes has no
virulence phenotype, which implies that their functions
might be redundant. Early studies of SPI2 T3SS effectors,
which primarily focused on determining their subcel-
lular localization in mammalian cells, revealed that
they might have specific targeting sequences that direct
localization to endosomal compartments, the Golgi
apparatus, the actin cytoskeleton and the microtubule
network33,155–157,159–162. This indicates that components
of these host-cell structures might be the intracellular
targets of the SPI2 T3SS.
Salmonella-induced tubular endosomes
In epithelial cells and interferon‑γ-primed macrophages,
the SPI2 T3SS induces the formation of long filamen-
tous membrane structures — Salmonella-induced fila-
ments (Sifs) — that originate from the SCV and extend
throughout the cell157,163 (FIG. 4). These structures are
LAMP1-positive and have a similar membrane composi-
tion to the SCV118. Sif formation requires microtubules,
but not the actin cytoskeleton, although these filaments
can be decorated with actin159. Possible mechanisms that
contribute to Sif formation include repetitive initiation
of vesicular budding from the SCV, in which fission
events are incomplete, or continuous fusion of endocytic
vesicles with the SCV, which would result in endosomal
tubulation or elongation of the SCV. Regardless of the
mechanism of Sif formation, the filaments may func-
tion to increase the size of the SCV to accommodate
bacterial replication during systemic infection. Sif
formation is dependent on the SPI2 T3SS effector SifA,
and to a lesser extent on SseF, SseG, SopD2 and PipB2
(Refs 91,164,165). However, it is a dynamic phenotype
that may also be modulated by other effectors. SifA was
recently shown to bind to the host cell protein SKIP (SifA
and kinesin interacting protein)166. The same authors
also found that, if SKIP was depleted by RNA interfer-
ence in S. typhimurium-infected cells, the cells failed to
form Sifs, suggesting that Sif formation requires SKIP.
In addition, Alto and colleagues167 identified SifA as a
member of the WxxxE (tryptophan (W)-variable (x)-x-
x-glutamate) family of bacterial effectors that function as
mimics of GTPases. As SifA contains a carboxy-terminal
Caax (cysteine (C)-aliphatic residue (a)-a-x) motif that

Prenylated
The post-translational addition
of lipid chains, such as farnesyl
or geranylgeranyl, to cysteine
residues in proteins that
contain a prenylation motif
called a CaaX box. This
process facilitates membrane
localization and/or protein–
protein interactions.
nature reviews | microbiology volume 6 | january 2008 | 61
is prenylated and S‑acylated by host-cell enzymes, similar
to GTPases, it is possible that SifA uses a GTPase-type
mechanism for membrane localization and, perhaps, Sif
formation168. Furthermore, the region of SKIP that inter-
acts with SifA is a pleckstrin homology domain, which
is commonly found in proteins that bind to signalling
lipids and other regulatory molecules, including those
that modulate Rho GTPases166.
The roles of PipB2, SopD2, SseF and SseG in Sif for-
mation are not entirely clear. S. typhimurium strains that
lack any of these effectors do not induce Sifs as efficiently
as wild-type S. typhimurium91,165,169. Instead, infection of
cultured cells with these mutants tends to lead to the for-
mation of ‘ pseudo-Sifs’, which extend from the SCV and
co-localize with effectors, but do not contain LAMP1.
This suggests that in the absence of these proteins the
ability to form Sifs — which is defined as being entirely
LAMP1-positive — is impaired and that induction of
Sifs involves multiple steps that are orchestrated by dif-
ferent effectors. Transient expression of PipB2 in mam-
malian cells induces the movement of LAMP1-positive
compartments to the cell periphery, which is probably
the result of its interaction with the plus-end-directed
microtubule motor kinesin165,170. This activity might
contribute to the outward extension of the SCV, which
would promote Sif formation. SopD2, which has homo­
logy to the SPI1 T3SS translocated effector SopD, has
also been shown to cooperate with SifA to induce Sifs,
but by an as yet unidentified mechanism33,91. SseF and
SseG promote the aggregation of endosomal vesicles
and recruit Golgi-derived exocytic vesicles to the SCV,
which suggests that salmonellae are able to usurp both
endocytic and exocytic cellular transport processes171–174.
However, it is not known how these activities directly
influence Sif formation.
The deletion of SseJ and SpvB can cause an increase
in Sif formation at later time points during the infection
of cultured cells, which suggests that these proteins have
Sif downregulatory functions175. Furthermore, these
effectors have virulence defects in the Nramp1-null ani-
mal model, which indicates that their activities contrib-
ute to systemic infection156,176. Although SpvB has not
been formally demonstrated to be an SPI2 T3SS effector,
some studies suggest that the SPI2 T3SS is required for
its activity in infected cells177,178. SpvB is an actin-specific
ADP-ribosyltransferase that promotes actin depolym-
erization176. SseJ has homology to glycerophospholipid
cholesterol acyl transferase enzymes, which can remove
acyl chains from phospholipids and transfer them to
cholesterol in a two-step deacylase-acyltranferase
reaction32,179. In fact, purified SseJ has deacylase activ-
ity in vitro, which has been shown to be required for
its virulence in mice180. It has been proposed that SseJ
and SifA have complementary roles in maintaining the
integrity of the SCV membrane, because sifA-mutant
S. typhimurium tends to lose the SCV membrane but
does not do so if SseJ is also lacking. This suggests that,
in the absence of SifA, SseJ could cause damage to the
SCV by its enzymatic activity. In addition, our labora-
tory has obtained evidence that SseJ has phospholipase
activity in vivo that might be localized to the endosomal
© 2008 Nature Publishing Group
REVIEWS
membrane, which has led us to propose that its most
likely function during S. typhimurium infection is to
cleave phospholipids from the SCV membrane (Megha,
M.B.O. and S.I.M., unpublished observations). The
cleavage of acyl chains from phospholipids could then
promote curvature of the membrane or produce discrete
lipid environments that serve as platforms for promot-
ing vesicle fusion and binding scaffolding proteins and
such activity could modulate Sif formation. However,
how these functions, and Sif formation in general, are
important for Salmonella-induced disease is currently
not understood. It is plausible that Sifs increase the size
of the SCV in a specific and directional fashion that
promotes bacterial replication and/or redirect nutri-
ent-rich organelles to the SCV. In this regard, the SPI2
T3SS could help salmonellae to replicate inside the
phagosome and gain important nutrients for growth,
but yet avoid some of the host-defence mechanisms and
inflammatory responses that would result from their
release into the cytoplasm.
Microtubule motors and movement of the SCV
As Sif formation requires microtubules — which have
been observed to accumulate and bundle around the
SCV — there has been an increased interest in under-
standing how microtubules and microtubule motors
contribute to the intracellular life cycle of salmonellae
and whether targeting vesicular trafficking along micro-
tubule networks in infected cells is indeed one of their
pathogenic strategies162 (FIG. 4). SifA has been reported to
downregulate the recruitment of the plus-end directed
microtubule motor kinesin to the SCV, which is medi-
ated by PipB2 (Refs 166,170). It does so by interacting
with SKIP, which, if in a complex with SifA, displaces
kinesin from the SCV166. This interference is thought to
be crucial for maintaining the integrity of the SCV. The
inhibitory activity of SifA on kinesin seems to be domi-
nant over the kinesin-recruiting activity of PipB2, which
is suggestive of a potential coordinate or temporal regu-
lation between the two effectors170. In addition, SseF and
SseG have been found to co-localize with microtubules
and cause microtubule bundling in S. typhimurium-
infected cells. In particular, SseF has been implicated
in recruiting the minus-end directed motor dynein to
the SCV162,173. Therefore, it would appear that the SCV
accumulates dynein, but not kinesin. However, as SifA
inhibits the interaction between Rab7 and its effector
RILP, a protein that is associated with the dynein motor
complex, it is plausible that SifA also prevents dynein
accumulation on the SCV181,182. Although these results
paint a paradoxical picture of the need to promote or
inhibit microtubule-motor accumulation on the SCV,
salmonellae might employ a carefully choreographed
combination of both the recruitment and inhibition
of both types of microtubule motors. For example, at
specific times during infection, the recruitment of
dynein could promote minus-end-directed movement
of the SCV to stabilize it near the nucleus or the Golgi
apparatus, where the SCV would have increased access
to trafficking vesicles that contain nutrients and mem-
brane, whereas at other times the recruitment of kinesin
REVIEWS
to the SCV could induce plus-end-directed movement
towards the cell periphery to promote the enlargement
of the SCV and/or elongation of Sifs.
Recent studies have also shown that the SCV
in infected cultured epithelial cells is localized in a
perinuclear position and that effectors from both the
SPI1- and the SPI2-encoded T3SSs are involved in this
process50,161,166,170,173,181. In particular, SifA, SseF, SseG
and SipA seem to control this phenomenon, because
the SCV containing S. typhimurium strains lacking any
of these effectors does not localize near the nucleus. As
the disruption of perinuclear localization of the SCV is
correlated with decreased bacterial replication, it has
been proposed that salmonellae require juxtanuclear
positioning for optimal growth. In fact, Salcedo and
Holden161 have shown that SCVs in close proximity
to the nucleus contain more bacteria on average than
SCVs further away from the nucleus. However, it
has not been directly demonstrated that non-nuclear
localization of the SCV, per se, is inhibitory to bacterial
replication. Rather, it seems more likely that the lack
of these effectors or disruption of vesicle trafficking
along the microtubule network correlates with defective
replication and that non-nuclear SCV positioning is a
by-product of this more direct effect. Furthermore, the
positioning of the SCV might be different in polarized
epithelial cells, in which movement of the vacuole in a
directional fashion, such as during transcytosis, could
be more physiologically relevant. As discussed above, it
is possible that positioning the SCV in close proximity
to the Golgi, or other organelles, has some advantages
for salmonellae in terms of acquiring nutrients, inhibit-
ing immune responses, prolonging the maturation of
intestinal epithelial cells and allowing more intracellular
time before epithelial shedding into the intestinal lumen.
All of these effects could then contribute to intestinal
colonization or the intracellular growth of salmonellae.
Actin polymerization around the SCV
SPI2 T3SS-dependent actin condensation around the
SCV has been observed, which suggests that this T3SS
is also involved in cytoskeletal modifications160,183 (FIG. 4).
In addition, the treatment of S. typhimurium-infected
cells with inhibitors of actin polymerization results in
decreased bacterial replication, which indicates that the
manipulation of the actin cytoskeleton is important for
bacterial replication183. Although the effectors that are
responsible for the recruitment of actin to the SCV have
not been identified, several have been shown to interact
with actin-binding proteins or to directly manipulate
actin polymerization. However, it is also possible that
SCV-associated actin polymerization is not mediated
by effectors, but is due to the insertion of the T3SS
translocon into the SCV membrane, which has also
been observed with the similar Yersinia spp. T3SSs184.
Indeed, most SPI2 T3SS effectors that have been shown to
interact with components of the actin cytoskeleton have
actin-inhibitory activities, which suggests that the most
likely goal of salmonellae is to reduce or reverse vacuole-
associated actin polymerization (VAP). For example,
SpvB has been shown to be involved in the disassembly
62 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
of VAP, presumably by ADP-ribosylating actin and
promoting actin depolymerization160,176. SspH2 has also
been reported to inhibit the rate of actin polymerization
in vitro160. In addition, SspH2 can interact with newly
polymerized actin, probably by binding to the actin-
binding proteins filamin and profilin, through its amino
and carboxy termini, respectively. Another effector, SseI
(also called SrfH), shares a high degree of identity with
the amino terminus of SspH2 and has been shown to
bind to polymerized actin, probably through filamin.
Although transiently expressed SspH2 and SseI are
associated with VAP during S. typhimurium infection,
they are not required for the formation of this structure.
Thus, unlike the clearly defined and temporally control-
led roles of the SPI1 T3SS effectors in manipulating the
actin cytoskeleton during invasion of epithelial cells, the
biological significance of the SPI2 T3SS-mediated actin
recruitment to the SCV, and the interaction of the effec-
tors of this T3SS with the actin network, remains to be
understood. It might be important for translocon stabil-
ity and translocation, thereby allowing the full effector
complement to be delivered consistently, and it could
explain the importance of this process to intracellular
replication183. Subsequently, as T3SS effectors seem to
decrease or remodel actin polymerization, there might
be a period in which actin removal is important, for
example, to decrease inflammatory responses or allow
fusion with, or remodelling of, the endosomal mem-
brane. Alternatively, actin removal could be essential for
the effectors to interact with the membrane surface, as
certain protein and phospholipid domains that are cov-
ered in polymerized actin may inhibit their binding to
important regions within the phagosome membrane.
Future perspectives
The study of the molecular basis of Salmonella spp.
pathogenesis in mammals has advanced greatly over
the past 15 years owing to the identification of many
of the key molecules and mechanisms in mammalian
model systems and the discovery of important innate
immune receptors and responses to the bacteria. For
example, important findings have been made in our
understanding of the mechanisms of entrance into non-
phagocytic cells, the way bacteria sense the intra­cellular
environment and remodel their surfaces, and the inflam-
matory responses to salmonellae. Concurrently, the
identification of innate immune receptors for bacterial
products and characterization of ligand binding by these
receptors has progressed greatly. We anticipate a fur-
ther explosion of information in the next decade about
the details of the intracellular lifestyle of salmonellae,
the function of effectors that are translocated across the
phagosome membrane and the response of the host to
the activity of these bacterial proteins. Studying how
salmonellae manipulate endosomal trafficking through
the endocytic pathway should lead to important obser-
vations about unknown mechanisms of endocytic traf-
ficking. In addition, how effectors function to bring
together mammalian protein components that may
not normally be together could offer important find-
ings with relevance for non-infectious diseases. The
 REVIEWS

study of salmonellae in more-resistant mouse models,
coupled with sophisticated imaging, should also lead to
a greater understanding of the pathophysiological role of
identified virulence mechanisms and the development
of chronic disease. This may have relevance for other dis-
eases at mucosal surfaces, such as inflammatory bowel
disease. The future decade may also take the field of
salmonellae pathogenesis and host responses beyond
model systems to the diversity of bacteria and hosts in
nature. Emerging technologies, such as whole-genome
sequencing, will allow us to investigate the diversity
of effectors that are used by the many pathogenic
Salmonella species, contributing to our view of host
specificity. Specific hypotheses will be generated by sta-
tistical association, which will then need to be tested in
model systems. In addition, the availability of human and
animal genotyping will lead to an increase in data about
the diversity of human and animal responses to infec-
tion with various salmonellae. Model systems in various
animals that have diverse innate immune modifications
will also need to be developed to further understand the
natural world in the context of our available tools and
to, ultimately, reach the stage in which the evolution of
microorganisms and the emergence of infectious diseases
can be observed in real time. Therefore, the study of sal-
monellae pathogenesis should yield a rich treasure trove
of information for those who are interested in microbial
pathogenesis, innate immunity, cell biology and genom-
ics, and should remain an important model system of
host–pathogen interactions for many years to come.

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SpvB. Mol. Microbiol. 39, 606–619 (2001).
197. Worley, M. J., Nieman, G. S., Geddes, K. & Heffron, F.
Salmonella typhimurium disseminates within its host
by manipulating the motility of infected cells. Proc.
Natl Acad. Sci. USA 103, 17915–17920 (2006).
198. Kujat Choy, S. L. et al. SseK1 and SseK2 are novel
translocated proteins of Salmonella enterica serovar
Typhimurium. Infect. Immun. 72, 5115–5125
(2004).
199. Rytkonen, A. et al. SseL, a Salmonella deubiquitinase
required for macrophage killing and virulence. Proc.
Natl Acad. Sci. USA 104, 3502–3507 (2007).
200. Geddes, K., Worley, M., Niemann, G. & Heffron, F.
Identification of new secreted effectors in Salmonella
enterica serovar Typhimurium. Infect. Immun. 73,
6260–6271 (2005).
201. Tsolis, R. M., Adams, L. G., Ficht, T. A. & Baumler, A. J.
Contribution of Salmonella typhimurium virulence
factors to diarrheal disease in calves. Infect. Immun.
67, 4879–4885 (1999).
Acknowledgments
We would like to thank the entire Salmonella pathogenesis
community for its work that made this Review possible. In
particular, we are grateful to members of the Miller labora-
tory, past and present, for their contributions to the ideas
that are presented here. We would also like to apologize to
those authors whose work was not cited owing to space limi-
tations. A.H. is supported by a Career Development Award
from the Northwest Regional Center of Excellence for
Biodefense and Emerging Infectious Diseases Research
(National Institute of Allergy and Infectious Diseases (NIAID)
grant U54 AI057141). M.B.O. is supported by the
Comprehensive Training in Inter-Disciplinary Oral Health
Research T32 grant DE07132. S.I.M. is supported by the
National Institutes of Health, NIAID grants R01 AI30479,
R01 AI048683 and U54 AI057141 for the Northwest
Regional Center of Excellence for Biodefense and Emerging
Infectious Diseases Research.
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=geneomeprj
Salmonella typhi | Salmonella typhimurium
All links are active in the online pdfF
 REVIEWS

An integrated model of the recognition

of Candida albicans by the innate
immune system
Mihai G. Netea*, Gordon D. Brown‡, Bart Jan Kullberg* and Neil A. R. Gow §
Abstract | The innate immune response was once considered to be a limited set of responses
that aimed to contain an infection by primitive ‘ingest and kill’ mechanisms, giving the host
time to mount a specific humoral and cellular immune response. In the mid‑1990s, however,
the discovery of Toll-like receptors heralded a revolution in our understanding of how
microorganisms are recognized by the innate immune system, and how this system is
activated. Several major classes of pathogen-recognition receptors have now been
described, each with specific abilities to recognize conserved bacterial structures. The
challenge ahead is to understand the level of complexity that underlies the response
that is triggered by pathogen recognition. In this Review, we use the fungal pathogen
Candida albicans as a model for the complex interaction that exists between the host
pattern-recognition systems and invading microbial pathogens.

Innate immune system
The suite of host responses to
microbial invaders that results
in rapid defence without
requiring prior stimulation.
*Department of Medicine
and Nijmegen University
Centre for Infectious
Diseases, Radboud University
Nijmegen Medical Centre,
Nijmegen, The Netherlands.
‡
Institute of Infectious
Diseases and Molecular
Medicine, Division of
Immunology, CLS, University
of Cape Town, Rondebosch
7925, South Africa.
§
School of Medical Sciences,
Institute of Medical Sciences,
University of Aberdeen,
Foresterhill, Aberdeen AB25
2ZD, UK.
Correspondence to M.G.N. &
N.A.R.G.
e-mails: m.netea@aig.umcn.nl;
n.gow@abdn.ac.uk
doi:10.1038/nrmicro1815
The clinical spectrum of Candida spp. infections ranges
from benign colonization of the skin and mucosal sur-
faces to mucocutaneous forms of candidiasis and sys-
temic infections (candidaemia and deep-seated organ
candidiasis). Despite the fact that fungal infections are
typically self-limiting, the number of life-threatening
systemic fungal infections has risen steadily over the
past three decades1, although this increase might now
be reaching a plateau2. The increased prevalence of fungi
as agents of disseminated infection has been restricted to
patients in whom surgical or chemotherapeutic interven-
tions and/or underlying immunological deficiencies have
allowed fungi to overwhelm the protective host defence
mechanisms2. In healthy and immunologically normal
individuals, the innate immune system is an efficient
sentinel that provides protection from the thousands of
fungal species that humans regularly encounter.
Candida albicans is a ubiquitous fungal organism that
often colonizes the skin and the mucosal surfaces of nor-
mal individuals, without causing disease. However, when
the normal host defence mechanisms are impaired (for
example, in patients who are undergoing chemotherapy
for malignancies, receiving immunosuppressants after
an organ transplant, or patients with AIDS), C. albicans
can become a pathogen. Candidaemia has an incidence
of between 1.1 and 24 cases per 100,000 individuals, and
an associated mortality of more than 30%3,4. It seems
unlikely that antifungal agents will have an impact on the
grim mortality statistics for systemic fungal infections
without the aid of new clinical approaches, such as com-
bining chemotherapy with immunotherapy5. However,
augmenting the ability of the immune system to elimi-
nate a pathogen requires a sophisticated understanding
of the molecular mechanisms that are involved in patho-
gen recognition and in the host immune response. In
this Review we describe the recent progress that has been
made in research into the mechanisms that are involved
in the recognition of fungal pathogens, and present
how this progress has changed our understanding of
the pathogenesis of infections in general, and invasive
candidiasis in particular.
Innate immunity and host defence
Until recently, little was known about the ways in which
neutrophils and macrophages, the major players in
innate immunity, recognized C. albicans as a pathogenic
microorganism, or how the fungal–leukocyte interaction
triggers an inflammatory response. The dogma that had
been blindly accepted over the past 50 years was that
although effective, innate immunity was non-specific
and “rather primitive and dumb” (Ref. 6). However, this
simplistic model, in which innate immunity performs
only simple ‘ingest and destroy’ tasks, could not explain
how innate immune cells recognize microbial pathogens

nature reviews | microbiology volume 6 | january 2008 | 67

© 2008 Nature Publishing Group
REVIEWS

Box 1 | The recognition of microorganisms by pattern-recognition receptors

The innate recognition of pathogens is achieved through germ-line-encoded receptors, the pattern-recognition
receptors (PRRs), which sense conserved chemical signatures called pathogen-associated molecular patterns (PAMPs).
Four major classes of PRRs have been identified.
• Toll-like receptors (TLRs) are cell-membrane-associated (TLR1, TLR2, TLR4, TLR5 and TLR6) or intracellular (TLR3, TLR7,
TLR8 and TLR9) receptors. Several TLRs have been implicated in the recognition of fungal components: TLR2 recognizes
phospholipomannan, TLR4 recognizes O‑linked mannans, TLR6 is involved in the recognition of zymosan and TLR9
detects fungal DNA.
• C-type lectin receptors (CLRs) are mainly membrane-bound receptors that recognize polysaccharide structures from
Candida albicans: dectin 1 recognizes β-glucans, whereas the macrophage mannose receptor (MR) and DC‑SIGN
recognize N‑linked mannans.
• Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and retinoic-acid-inducible gene I (RIGI)
receptors are intracellular receptors for bacterial peptidoglycans and viral nucleic acids. So far, no studies have
documented the involvement of NLRs or RIGI receptors in the recognition of fungi.
• The blueprint for the structure of PRRs is represented by the extracellular pathogen-recognition domain (the Leu-rich
repeat (LRR) domain in TLRs and the C‑type lectin domain (CLD) in CLRs). The intracellular signalling domain (the TLR-
interleukin 1 receptor, TIR domain) of TLRs and the immunoreceptor Tyr-based activation-like motif (ITAM) from some
CLRs, such as dectin 1, ultimately induce the intracellular signals responsible for the functional activity of the receptor.
The four major classes of PRRs discussed here are restricted to families of mammalian molecules with a PRR
function at a cellular level that have a proven signalling pathway that leads to activation of the innate host response.
There are other classes of molecules with less well established functions as PRRs. (For example, peptidoglycan-
recognition proteins (PGRPs), which recognize peptidoglycans in insect and mammalian cells. Although PGRPs
induce defensins in Drosophila melanogaster, their function in mammalian cells has not yet been established.) Some
circulating proteins that bind bacterial structures (for example, pentraxins and mannose-binding lectin (MBL)), are
also considered by some to be PRRs.

Dendritic cells
‘Professional’ antigen-
presenting cells that are found
in the T-cell areas of lymphoid
tissues and as minor cellular
components in most tissues.
They have a branched or
dendritic morphology and are
important stimulators of T-cell
responses.
as ‘non-self ’, or why different responses are triggered by
different classes of microorganisms. Only in the past
decade has it become clear that the innate immune
system not only specifically recognizes various classes
of microorganisms, it also initiates and modulates the
subsequent adaptive responses that are delivered by T-
cells and B cells through their interactions with antigen-
presenting cells (especially dendritic cells (DCs))7. The
tasks of recognizing an invading pathogen and acti-
vating the host response are accomplished by pattern-
recognition receptors (PRRs), which recognize conserved
microbial chemical signatures called pathogen-associated
molecular patterns (PAMPs) (BOX 1).
The fungal cell wall
The fungal cell wall is an essential organelle that main-
tains the viability of fungal cells. The regulation of fungal
cell wall biosynthesis and glycosylation has been exten-
sively reviewed elsewhere8–10, but a basic outline of the
components of the cell wall is necessary to understand
how it is recognized by the host immune system. To be
strong, yet plastic, fungal cell walls combine skeletal and
matrix components in a way that resembles the engineer-
ing principles that are involved in constructing elaborate
structures, such as reinforced concrete buildings, that
are made of mesh and mortar. The cells of the innate
immune system recognize elements of both the skeletal
and matrix components of the C. albicans cell wall.
The skeletal component of the cell wall of the major-
ity of fungal pathogens, including C. albicans, is based
on a core structure of β-(1,3)-glucan covalently linked
to β-(1,6)-glucan and chitin (a β-(1,4)-linked polymer of
N‑acetylglucosamine (GlcNAc)) (FIG.1). These polymers
form hydrogen bonds between adjacent polysaccharide
chains to form a tough three-dimensional network of
microfibrils. Most models suggest that the skeletal com-
ponents of the cell wall are found close to the cell mem-
brane in an inner layer, although some chitin and glucan
can be present throughout the thickness of the wall. In
budding yeast cells, a scar is left on the mother cell after
separation of the bud, and at this site the components
of the inner layers of the cell wall, such as chitin and
β-(1,3)-glucan, can become exposed at the surface11.
In addition to the glucan and chitin skeleton, the
C. albicans cell wall contains a matrix that mainly com-
prises glycosylated proteins. In C. albicans, the major
class of cell wall proteins are glycosylphosphatidylinositol
(GPI)-anchor-dependent cell wall proteins (GPI-CWPs),
which are attached through a GPI remnant to β-(1,3)-
glucan or chitin by a highly branched β-(1,6)-glucan
linker. The CWPs are normally highly glycosylated with
mannose-containing polysaccharides (sometimes called
mannan), and carbohydrates can account for up to 90%
of their molecular mass. Many CWPs have a lollipop
structure with a globular domain that is presented to
the outside of the cell and a Ser/Thr-rich polypeptide
stem-like domain that is stabilized in the cell wall by
O‑linked mannan side chains 8. These ether-linked
O‑mannans are relatively short, linear polysaccharides
that, in C. albicans, consist of one to five mannose (Man)
sugars that are almost exclusively α-(1,2)-linked (FIG. 1).
N‑mannan consists of a core Man8GlcNAc2 trianten-
nary complex to which a highly branched structure is
attached, comprising up to 150 mannose sugars arranged
as an α-(1,6)-linked backbone with side chains of α-
(1,2)-, α-(1,3)-mannose and phosphomannan9 (FIG. 1).
The mannan structures define many of the serotypes
of Candida spp.12 For example, in serotype B strains

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Cytokines
Biologically active molecules
that are released by cells and
that affect the function of other
cells.
Fcg receptor
A surface molecule on various
cells that binds to the Fc
regions of immunoglobulins,
thereby initiating effector
functions.
T helper 1
An immune response that is
characterized by a subset of
helper T-cells that secrete a
particular set of cytokines,
including interleukin 2,
interferon-γ and TNFα, the
main function of which is to
stimulate phagocytosis-
mediated defences against
intracellular pathogens.
C-type lectin
C-type lectins are largely
calcium-dependent animal
lectins that are carbohydrate-
binding proteins. The binding
activity of C-type lectins is
based on the structure of the
carbohydrate-recognition
domain, which is highly
conserved in this family.
nature reviews | microbiology volume 6 | january 2008 | 69
Mannan N-acetylglucosamine
Cell wall protein α-(1,3)-mannose
β-(1,6)-glucan α-(1,2)-mannose
β-(1,3)-glucan α-(1,6)-mannose
Chitin β-(1,2)-mannose
β-(1,4)-mannose
β-(1,6)-glucose
β-(1,3)-glucose
Figure 1 | The structure of the Candida albicans cell wall. The schematic shows the major components of the cell wall
and their distributions. β-(1,3)-glucan and chitin (poly-β-(1,4)-N‑acetylglucosamine) are the main
and these are located towards the inside of the cell wall. The outer layer is enriched with cell wall proteins (CWP) that are
attached to this skeleton mainly via glycosylphosphatidylinositol remnants to β-(1,6)-glucan or, for mannoproteins with
internal repeat domains (Pir-CWP), via alkali-sensitive linkages to β-(1,3)-glucan. The insets show the structure of the
glucan and mannan components.
β-(1,2)-mannose is found exclusively in the phospho-
mannan, whereas in serotype A strains β-(1,2)-mannose
also occurs in the acid-stable side-chain fraction9.
Fungal recognition
Owing to the localization of mannoproteins and man-
nans in the outermost part of the cell wall, mannan
detection would be expected to be one of the first steps in
the recognition of C. albicans by the host innate immune
system. However, the presence of β‑glucans and chitin,
especially at the level of the bud scar, is also likely to
influence the recognition of C. albicans by leukocytes.
So how is the recognition of the C. albicans cell surface
achieved by the immune system?
The main cells of the host innate immune response
that recognize invading pathogens are monocytes and
neutrophils in the circulation, together with macro-
phages in infected tissues. Monocytes express high levels
of Toll-like receptors (TLRs) on their cell membranes, as
well as moderate levels of lectin receptors (LRs). During
differentiation into macrophages, they retain expression
of TLRs while strongly upregulating their expression of
LRs. Important differences in the expression of several
receptors have also been reported between resident and
inflammatory macrophages, as the expression of PRRs
can be modified by cytokines or microbial products. DCs,
which are crucial for antigen processing and presenta-
tion, also express most of the PRRs that are important
for the recognition of fungal pathogens. Neutrophils
show moderate expression of TLRs and strongly express
phagocytic receptors such as complement receptor 3
(CR3) and Fcγ receptors (FcγRs), but the expression of
PRRs on T cells is much more restricted (FIG. 2). The
mosaic of PRRs that is expressed by each of these cell
© 2008 Nature Publishing Group
REVIEWS
P
n
NH O
Asn X Ser/Thr Ser/Thr
Pir-CWP
CWP
structural
Nature Reviewscomponents,
| Microbiology
types ultimately determines the type of response that is
initiated following recognition of C. albicans.
Mannans and mannoproteins. Both mannans and man-
noproteins from the C. albicans cell wall have important
immunostimulatory activities, ranging from stimulation
of cytokine production13,14 to induction of DC matura-
tion15 and T-cell immunity16,17. Mannoproteins induce
mainly T helper 1 (TH1)-type cytokine profiles, which
have protective effects against disseminated C. albicans
infection18.
The first receptor on the surface of macrophages to
be described as a mannan receptor was the C‑type-lectin
mannose receptor (MR)19,20. The MR recognizes oligosac-
charides that terminate in mannose, fucose and GlcNAc21,
and this binding is mediated by carbohydrate-recognition
domains (CRDs) 4 to 8 in the extracellular region of
the receptor22. In vitro studies have shown that the MR
preferentially recognizes α-linked oligomannoses with
branched, rather than linear, structures23, and this was
supported by data that demonstrated that in mono-
cytes and macrophages, the MR recognizes branched
N‑bound mannans from C. albicans24. By contrast,
recognition of the shorter linear structures of O‑bound
mannan is performed by TLR4 (Ref. 24), and results in
cytokine production25. Interestingly, TLR4 stimulation
is lost during the germination of yeast into hyphae,
which leads to a loss of interferon-γ (IFNγ) production
capacity26. On DCs, however, the binding of C. albicans
mannans is mediated through the MR and DC‑SIGN
(DC‑SIGN is a receptor that is specifically expressed on
the cell membrane of DCs)27.
The α-linked mannose structures on the surface
of C. albicans are recognized by the MR, TLR4 and
REVIEWS

Monocyte suggests that β-glucans are exposed on the cell surface35,

TLR4 although possibly restricted to specific regions, such as
Dectin 1
bud scars11. β‑glucans can stimulate leukocytes in vitro,
TLR2 which induces cytotoxic and antimicrobial activities as
MR
TLR6 well as the production of pro-inflammatory mediators,
Neutrophil Macrophage cytokines and chemokines36. β‑glucans are released into
TLR9
TLR2 TLR6 the circulation during systemic fungal infections37.
TLR2 TLR4 The recognition of β‑glucans has primarily been
Dectin 2 Dectin 1
ascribed to two receptors, CR3 and, more recently,
dectin 1. Although other β‑glucan receptors have been
CR3 Galectin 3 TLR9 described, including lactosylceramide and scavenger
MR
receptors, the physiological role of these receptors is
FcγR Dectin 1 TLR4 still unclear. CR3 is a widely expressed β2-integrin that
CR3
FcγR recognizes several endogenous and exogenous ligands,
Dendritic cell
CD4
pathogens that have been opsonized by iC3b (the inac-
TLR2 Dectin 2 tivated form of complement component C3b) and carbo-
TLR2
hydrates, including β‑glucans. Carbohydrate recognition
DC- Dectin 1
is mediated by a lectin domain38,39, which is distinct from
SIGN
TLR9 TLR9 the normal ligand-binding site (the I domain) of CR3
MR
TLR4 (Ref. 39). The lectin domain mediates recognition of both
TLR4
the yeast and hyphal forms of C. albicans40,41, as well as
FcγR
CR3 several other fungi. Recognition by CR3 does not trigger
Figure 2 | Cell populations and pattern-recognition receptors involved in
protective host responses, such as the respiratory burst42,
Candida albicans recognition. The main populations involved Nature Reviews
in the | Microbiology
recognition and can repress pro-inflammatory signals43,44.
of C. albicans during the innate immune response are the monocytes, neutrophils and Dectin 1 is a myeloid-expressed transmembrane
macrophages. Dendritic cells are crucial for processing of, and antigen presentation to, receptor and possesses a single extracellular, non-
T cells, and thus to activation of specific immunity. The differential expression of pattern- classical C‑type-lectin-like domain that specifically rec-
recognition receptors by these cell types is shown. CR3, complement receptor 3; FcγR, ognizes β-(1,3)-glucans. Dectin 1 can recognize several
Fcγ receptor; MR, mannose receptor; TLR, Toll-like receptors. fungi, including C. albicans yeast, although it does not
appear to recognize C. albicans hyphae11,45. The cyto-
plasmic tail of dectin 1 contains an immunoreceptor
DC‑SIGN. By contrast, β-(1,2)-mannosides, which are tyrosine-based activation-like motif (ITAM), which can
present in the acid-stable and acid-labile components mediate various protective responses through spleen
of mannoproteins and in phospholipomannan (PLM), tyrosine kinase and caspase recruitment domain pro-
are recognized by other mechanisms. On the one hand, tein 9 (Syk–CARD9)-dependent pathways, such as the
PLM reportedly stimulates cytokine production through stimulation of interleukin 2 (IL-2) and IL‑10 (Ref. 46),
TLR2 (Ref. 28). On the other hand, it has been suggested IL‑6 (Ref. 47), and IL‑17 production48. Although Syk-
that recognition of the acid-labile β-(1,2)-mannosides is dependent signalling from dectin 1 is sufficient for
redundant in human monocytes24, although it might play a these responses, stimulation of the mitogen-activated
role in tissue macrophages, particularly in the gut mucosa. protein kinase (MAPK) and nuclear factor (NF)-κB
In this respect, a recent study has shown that galectin 3 pathways, with subsequent production of pro-inflam-
on the surface of murine macrophages can discriminate matory cytokines, such as tumour necrosis factor (TNF),
between pathogenic C. albicans and non-pathogenic requires collaborative signalling with the TLR2 recep-
Saccharomyces cerevisiae, and that an association between tor49,50. The precise nature of the TLR2 ligand from C.
galectin 3 and TLR2 is involved in this process29. albicans has not yet been completely elucidated, although
Another lectin family member, dectin 2, has also PLM might be recognized by TLR2 and TLR6 (Ref. 28).
been described to function as a receptor for C. albicans It has recently been suggested that phagocytosis of
mannans30, although this interaction is less well charac- C. albicans by neutrophils can be mediated by the minor
terized. Dectin 2 is mainly expressed on myeloid cells cell wall component β-(1,6)-glucan51. Beads coated with
and maturing inflammatory monocytes31. Owing to its β-(1,6)-glucan are ingested by neutrophils, and the
short intracytoplasmic tail, dectin 2 must interact with phagocytosis of yeast cells treated with β-(1,6)-glucanase
the FcγR to induce intracellular signals and, interest- is reduced. This recognition appears to be mediated by
Chemokines
ingly, seems to be mainly involved in the recognition of CR3 following opsonization by C3d fragments that bind
Small, inducibly secreted
proteins that induce the C. albicans hyphae32. Dectin 2 is encoded by a different β-(1,6)-glucan.
activation and migration of gene cluster to the β-glucan receptor dectin 1 (see below)
lymphocytes. and does not recognize β‑glucans32; rather, dectin 2 has Other C. albicans components. In addition to manno­
binding specificity for mannose33. proteins, mannans and β‑glucans, other structures of
Complement C. albicans can also be recognized as fungal PAMPs.
A part of the innate immune
system that comprises serum
β-glucans. The C. albicans cell wall consists of approxi- Chitin, a less studied polysaccharide component of the
proteins that can protect mately 60% β-glucan34. Although initially thought to be C. albicans cell wall, is a β-(1,4)-linked homopolymer of
against infection. buried beneath the mannoprotein layer, recent evidence GlcNAc that forms antiparallel hydrogen-bonded chains

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Type I interferons
Interferons that are rapidly
induced by virus replication, as
well as by some bacterial and
fungal infections.
TH2
A type of activated T helper
cell that participates in
phagocytosis-independent
responses and that
downregulates pro-
inflammatory responses that
are induced by TH1 cells. TH2
cells secrete interleukin 4
(IL-4), IL-5, IL-6 and IL-10.
Regulatory T-cell
A small population of CD4+
T-cells that expresses the
transcription factor forkhead
box P3 and that has
suppressor activity towards
other T-cells either by cell–cell
contact or cytokine release.
Zymosan
Particulate preparation of
S. cerevisiae cell walls that is
rich in β-glucans and mannan
and that can be used to
activate the innate immune
system.
nature reviews | microbiology volume 6 | january 2008 | 71
called microfibrils52. Recently, it has been demonstrated
that chitin induces recruitment of immune cells that
mainly release IL‑4 and IL‑13 (Ref. 53). Little is known
about the recognition pathways of chitin and its role dur-
ing C. albicans infections, and no recognition receptors
for chitin have yet been described.
Bacterial and fungal DNA are poorly methylated, in
contrast to mammalian DNA. This difference has been
proposed to be instrumental in the recognition of non-
self DNA by TLR9 (Ref. 54). Although the recognition of
fungal DNA has not been formally demonstrated, the
involvement of TLR9 in the recognition of C. albicans is
supported by the observation that cytokine production
from CD4+ T-cells from TLR9–/– mice is skewed (higher
IL‑4, lower IFNγ) compared to cytokine production
from CD4+ T cells from wild-type mice, upon challenge
with C. albicans yeast55. As yet, no studies have investi-
gated the possible role of RNA recognition systems (that
is, TLR3, and TLR7 and TLR8) in the host response to
C. albicans infection.
Activation of host defence by PRRs
At first glance, ligation of the various host immune
receptors by C. albicans PAMPs appears to lead to a
set of standard, and possibly redundant, pathways that
stimulate cytokine production, phagocytosis and fun-
gal killing. However, this early model of a ‘standard’
PRR response has been refined over the past few years.
Various PRRs enable the innate immune system not
only to recognize specific PAMPs, but also to specifi-
cally modulate the response that follows. In addition, by
inducing specific cytokine profiles, PRRs bring a certain
degree of specificity to the innate response.
C. albicans uptake. Phagocytosis of C. albicans is medi-
ated by the concerted action of several opsonic and non-
opsonic receptors. Complement binding and activation
is mediated by the alternative pathway, and complement
activation is mainly important for the chemotaxis and
opsonization of C. albicans, but not for C. albicans lysis,
which is prevented by the thick and complex cell wall56.
Furthermore, although mannose-binding lectin does
bind and recognize C. albicans, the lectin pathway of
complement activation probably plays only a minor role
in C. albicans uptake57.
Several membrane-bound receptors contribute to
the phagocytosis of C. albicans, among which dectin 1
(Ref. 58), the MR59,60 and DC‑SIGN27 have been directly
shown to mediate uptake of fungal particles in trans-
fected cell systems, although the ability of the MR to
mediate phagocytosis has recently been questioned61.
TLRs do not mediate fungal uptake, but they might be
involved in directing the subsequent maturation of the
phagosome62 and the presentation of antigens, although
this is still controversial62.
C. albicans killing. Following uptake, killing of
C. albicans occurs through both oxidative and non-
oxidative mechanisms63. Although the receptors that are
involved in triggering these events are largely unknown,
dectin 1 induces the respiratory burst in response to
© 2008 Nature Publishing Group
REVIEWS
fungi, an activity that can be enhanced by TLR signal-
ling50,64. The respiratory burst is an essential antifungal
effector mechanism that results in the production of
toxic oxidants65,66 and in the activation of granule pro-
teases that can kill C. albicans65–67. Killing of C. albicans
also occurs extracellularly, through the as-yet-undefined
actions of PRRs such as galectin 3 (Ref. 68).
Cytokine induction. More is known about the receptors
that are involved in the induction of cytokine production
by C. albicans. At least four TLRs (TLR2, TLR4, TLR6
and TLR9) are involved in triggering these responses.
Upon recognition of microbial structures, TLRs activate
either the NF‑κB or MAPK pathway, which lead to the
stimulation of pro-inflammatory cytokine produc-
tion69. The balance between the signals that are induced
by TLR2 and TLR4 seems to have a crucial role in the
regulation of the immune response. TLR4 can strongly
stimulate pro-inflammatory cytokines through two path-
ways: one involves the myeloid differentiation primary
response gene 88 (MyD88)–Mal-mediated induction
of the NF‑κB-dependent release of pro-inflammatory
cytokines and chemokines; the other involves the inter-
feron regulatory factor 3 (IRF3)-dependent release of
type I interferons70, which induce the secondary production
of TH1-type cytokines such as IFNγ 26,55,71.
By contrast, several studies have demonstrated that
although TLR2 ligation can induce pro-inflammatory
cytokines, this effect is weaker than that mediated by
TLR4 ligation72. Moreover, TLR2 ligands fail to induce
the release of IL‑12 and TH1-type IFNγ, thus promoting
conditions that are favourable for TH2- or regulatory T cell
(TReg)-type responses73. Stabilization of the transcription
factor c‑Fos, which is a suppressor of IL‑12 expression,
is an important step in this process74. In support of
this, a recent in vitro study has reported that zymosan
induces the development of a tolerigenic DC population
through TLR2 and dectin 1 (Ref. 75). The induction of
this tolerigenic cytokine profile depends on extracel-
lular signal related kinase (ERK)/MAPK phosphoryla-
tion, a mechanism that is distinct from the p38/Jun
N‑terminal kinase (JNK) pathway that is induced by
TLR4 stimulation75,76. Also, in vivo studies have shown
that knockout mice that lack TLR2 are more resistant
to disseminated candidiasis, and this is accompanied
by a TH1 bias55,77. C. albicans induces immunosuppres-
sion through TLR2-mediated IL‑10 release, and this
leads to the generation of CD4+CD25+ TReg cells with
immunosuppressive potential77,78. Similar data have
been reported in models of Schistosoma mansoni and
Borrelia burgdorferi infection79,80.
The role of TLR6 and TLR9 in the induction of
cytokines is less prominent. Although TLR2–TLR6
heterodimers are involved in the recognition of
zymosan81, cytokine production is only moderately
reduced in TLR6–/– macrophages that are stimulated
with C. albicans, and this does not result in increased
susceptibility to disseminated candidiasis82. Similar to
TLR2, the absence of TLR9 also results in a slight shift
of the cytokine production that is induced by C. albicans
towards a more anti-inflammatory profile55.
REVIEWS
Tetraspanin
A family of transmembrane
proteins that have four
transmembrane domains and
two extracellular domains of
different sizes. The function of
tetraspanins is unclear, but
they seem to interact with
many other transmembrane
proteins and form large
multimeric protein networks.
TH17 response
The TH17 subpopulation are
T-cells that release mainly
IL-17, which has both
neutrophil chemotactic activity
and the potential to promote
autoimmunity.
72 | january 2008 | volume 6 www.nature.com/reviews/micro
Of the non-TLR receptors, dectin 1, dectin 2 and
the MR have been implicated in initiating responses to
C. albicans. Not much is known about dectin 2, but this
receptor does preferentially bind C. albicans hyphae
and induces the production of TNFα and IL-1 receptor
antagonist (IL-1Ra)32. Dectin 1 induces the production
of numerous cytokines and chemokines in response
to fungi, including TNFα, macrophage inflammatory
protein 2 (MIP2), macrophage inflammatory protein 1α
(MIP1α), granulocyte–macrophage colony-stimulating
factor (GM-CSF), granulocyte colony-stimulating fac-
tor (G-CSF), IL‑10, IL‑2, IL‑1α, IL‑1β, IL‑23 and IL‑6
(Refs 46,50,83–86). Whereas the production of IL‑10,
IL‑6 and IL‑2 can be mediated by dectin 1 directly87, the
induction of pro-inflammatory cytokines and chem-
okines requires collaborative signalling with TLR2
(Refs 50,83), although evidence suggests that dectin 1 is
sufficient for these responses in certain cell types, such
as alveolar macrophages85. By contrast, the interaction of
dectin 1 with the tetraspanin CD37 seems to inhibit the
function of dectin 1 and stimulation of IL‑6 (Ref. 88).
The intracellular pathways that are activated by dec-
tin 1 for the induction of cytokines involve the recruit-
ment of Syk46 and downstream signalling via CARD9
(Ref. 89). The dectin 1–Syk–CARD9 pathway induces
DC maturation and directs TH17 responses that are inde-
pendent of the interaction with TLRs48, and this effect
has been particularly linked with hyphal stimulation of
DCs90. The role of IL‑17 in the host response to dissemi-
nated candidiasis has been suggested to be protective, by
inducing neutrophil recruitment91, whereas it exerts a
predominantly inflammatory pathological effect in gas-
tric C. albicans infections92. In addition to its mediation
of the dectin 1–Syk pathway, CARD9 is also a central
adaptor molecule that mediates the pro-inflammatory
signals that are induced by other classes of receptors,
such as the nucleotide-binding oligomerization domain
(NOD)-like receptors93, ITAM-associated receptors and
possibly the TLRs94. Recently it has also been suggested
that TRIF-dependent pathways modulate the balance
between deleterious TH17 responses and protective TReg
cells in mice with gastric candidiasis95.
The receptors and intracellular pathways that are
involved in the recognition of C. albicans by leukocytes
are depicted in FIG. 3. Although all of these pathways have
been demonstrated to be involved in the recognition of
fungal components, it has not been verified that identical
mechanisms are responsible for the recognition of intact,
live C. albicans cells. For example, zymosan has been used
extensively for immune stimulation, but the relationship
between the response to zymosan and the response to
native fungal cells has not yet been clarified. The spe-
cificity of the antifungal response is evident in differences
between the kinetics of the various immunological effects
and in the magnitude of each of these responses.
Epithelial and endothelial surfaces also have PRRs
that recognize surface PAMPs of invading and colonizing
microorganisms such as C. albicans. Studies of the role of
epithelial cell immunity have been significantly enhanced
by the availability of model epithelial and endothelial tis-
sues in which a cell line of keratinocytes is grown on the
© 2008 Nature Publishing Group
surface of a polycarbonate support membrane. Immune
cells such as polymorphonuclear leukocytes (PMNs)
can be added to such ex vivo reconstituted human epi-
thelial (RHE) tissue cultures along with an inoculum of
fungal cells. The expression of TLRs in RHE models is
similar to the TLR expression profile in vivo96. Using such
approaches, PMNs have been shown to be active in the
innate immune response at these primary barriers to sys-
temic invasion97, and differences have been noted between
the local immunity of the oral and vaginal mucosae97,98. For
example, PMNs enhance an inflammatory TH1 response
in the oral epithelia, which results in the induction of pro-
inflammatory cytokines, such as IFNγ and TNFα, effec-
tively mimicking the situation that is observed in vivo97.
Vaginal epithelial cells express TLR2 and TLR4 in vivo,
and the presence of heat-inactivated C. albicans cells and
zymosan induces these cells to secrete inflammatory
cytokines, chemokines and to produce β-defensin 2 — an
antimicrobial compound that also induces PMN chemo-
taxis99. Work to characterize fungal PAMPs and PRRs that
induce local innate responses is ongoing. For example, it
has recently been shown that human epithelial TLR4 is
directly involved in the PMN-dependent protection of
oral mucosa96.
In vivo models in TLR- and LR‑deficient mice
In vitro stimulation models that use either cell lines or
primary cells have suggested specific roles for the various
PRRs in the recognition of C. albicans, as detailed above.
However, whether these pathways are indeed important
for the antifungal host response in vivo can only be
demonstrated in experimental models of infections in
genetically modified mice that lack particular receptors,
or in patients with specific immune defects.
Since the first observation that TLRs recognize
C. albicans and activate the innate immune response74,
additional studies have confirmed the important role
that TLRs have in the recognition of C. albicans and the
anti-candidal host response. A global role for TLRs in
defence against disseminated candidiasis was demon-
strated by the increased susceptibility of MyD88–/– mice
to C. albicans infection55,100,101. A subsequent study
reported increased susceptibility of TLR2–/– mice to
disseminated candidiasis, and this effect was attributed
to decreased production of TNF and chemokines102.
However, the increased susceptibility of TLR2–/– mice
to infection with C. albicans was not confirmed in two
other studies, which found reduced mortality and a
decreased fungal burden in TLR2–/– mice, accompanied
by decreased production of IL‑10 and increased pro-
duction of IL‑12 and IFNγ 55,77. An additional in vitro
study also confirmed the increased ability of macro-
phages from TLR2–/– mice to contain C. albicans103.
These latter studies55,77,103 suggest that TLR2, by induc-
ing mainly an anti-inflammatory response, inhibits
the innate immune response to C. albicans infections
in certain circumstances.
It has also been suggested that TLR4 is involved in
the recognition of C. albicans24,25,71, and TLR4-defective
C3H/HeJ mice have been reported to be more suscepti-
ble to disseminated candidiasis71. However, other studies
 REVIEWS

Monocytes/macrophages Dendritic cells

Mannan β-Glucans β-Glucans Mannan Mannan Mannan β-Glucans Mannan Mannan
(O-linked) PLM? β-Mannosides (N-linked) Chitin (N-linked) (O-linked) PLM? β-Glucans (N-linked) (N-linked)

TLR4 TLR6 TLR2 + Galectin 3 Dectin 1 MR Chitin R? Dectin 2 FcγR TLR4 TLR2 Dectin 1 MR DC-
SIGN

Mal Mal Syk TRIF Mal Mal Syk

MyD88

MyD88

MyD88
+

MyD88
TRAM
+

IκB ERK IκB CARD9 IκB p38/JNK ERK CARD9

NF-κB NF-κB NF-κB

NF-κB
NF-κB NF-κB NF-AT IRF3 NF-κB c-Fos

IL-8 TGFβ TNF IL-10 TNF IL-4 TNFα Type I TNF IL-12 TGFβ IL-23 TNF IL-10
TNF IL-10 IL-2 IL-1β IFN IFNγ IL-10 IL-6 IL-1β

TReg TH2 TH1 TH1 TReg TH17

Figure 3 | Recognition of Candida albicans at the membrane level. Recognition of C. albicans at the level of the cell
membrane is mediated by TLRs and LRs. TLR4 induces mainly pro-inflammatory signals in monocytic cell types
(monocytes, macrophages and DCs) through the MyD88–Mal-mediated NF‑κB and MAPK pathways, Nature Reviews | Microbiology
while stimulating TH1
responses through IRF3-dependent mechanisms mainly in plasmacytoid DCs. TLR2 stimulates the production of moderate
amounts of pro-inflammatory cytokines and strong IL‑10 and TGFβ responses. On the one hand, this leads to the induction
of a tolerant phenotype in DCs, through an ERK/MAPK-dependent mechanism121. On the other hand, in monocytes and
macrophages it induces TGFb and IL-10, and subsequent proliferation of TReg cells and immunosuppression78. The pro-
inflammatory effects of TLR2 can be amplified by dectin 1 and galectin 3 — the latter especially in macrophages. In
addition to amplifying the effects of TLR2, the non-classical lectin-like receptor dectin 1 induces IL‑2, IL‑10 and TH17
responses through a Syk–CARD9 cascade, independently of its interaction with TLR2. The classical lectin-like receptor, the
MR, induces pro-inflammatory effects in monocytes and macrophages, whereas chitin-dependent stimulation of these
cells induces mainly TH2 responses122, although this effect has yet to be demonstrated for C. albicans, and the identity of
the chitin receptor is unknown. Other less well characterized pathways include stimulation of TNF and IL‑1Ra by dectin 2,
while engagement of DC-SIGN in DCs induces production of the immunosuppressive cytokine IL‑10 . CARD9, caspase
recruitment domain protein 9; DC, dendritic cell; ERK, extracellular signal related kinase; FcγR, Fcγ receptor; IL,
interleukin; IL‑1Ra, interleukin‑1 receptor antagonist; IFNγ, interferon-γ; IRF3, interferon regulatory factor 3; JNK, Jun
N‑terminal kinase; LR, lectin receptor; MAPK, mitogen-activated protein kinase; MR, mannose receptor; MyD88, myeloid
differentiation primary response gene 88; NF‑κB, nuclear factor κB; PLM, phospholipomannan; Syk, spleen tyrosine kinase;
TGFβ, transforming growth factor-β; TH, T helper; TLR, Toll-like receptor; TNF, tumour necrosis factor; TReg, regulatory T-cell.

have observed variable results, with TLR4–/– mice being
more susceptible in models of intragastric infection or
intravenous re-infection 55, not different from wild-
type animals in models of intravenous infection with
C. albicans yeast104, or even surviving longer in a model
of intravenous infection with C. albicans hyphae55. The
differences between the experimental models and/or
the C. albicans strains used are thought to be responsi-
ble for these differences.
Although no increased susceptibility of TLR9–/– mice
to disseminated candidiasis has been observed, the
fungal burden in the organs of TLR9-deficient animals
tends to be lower than in control mice55. Although
pro-inflammatory cytokine production was slightly
lower in TLR9–/– macrophages that had been stimulated
with C. albicans, this mild difference did not result in
a deleterious outcome of infection, probably owing to
compensatory pathways that are mediated either by
other TLRs or by LR‑dependent mechanisms.
The study of the role of LRs in the in vivo response
to C. albicans infections is still in its infancy. Recently, it
has been shown that dectin 1–/– mice are more susceptible
to C. albicans infection, and infection results in increased
fungal growth in the organs and accelerated death105.
This is consistent with the increased susceptibility of
CARD9–/– mice to disseminated candidiasis89. Notably,
all of the major components of antifungal defence
— cytokine release, phagocyte recruitment, phagocytosis
and microbial killing — are impaired in the knockout
mice, which suggests an important role for dectin-1-
mediated recognition of β-glucans for anti-candidal
defence in vivo105. However, these results are at odds with

nature reviews | microbiology volume 6 | january 2008 | 73

© 2008 Nature Publishing Group
REVIEWS

a b c
C. albicans C. albicans C. albicans
yeast hyphae yeast

TLR2 TLR4 Dectin 1 CR3 FcγR TLR4

Mal Mal Mal

MyD88

MyD88

MyD88
Syk

β-Glucans shielded from

dectin 1 recognition
ERK NF-κB NF-κB

c-Fos

Anti-inflammatory Pro-inflammatory Pro-inflammatory

signals signals signals

Figure 4 | Candida albicans mechanisms to escape the innate response using pattern-recognition receptors.
Nature Reviews
| Microbiology
a | The balance between the signals that are induced by Toll-like receptor 2 (TLR2) and TLR4 seems to have a crucial role in
the regulation of the immune response. TLR2-mediated signals are mainly anti-inflammatory and can be deleterious when
activated prematurely or excessively. TLR4-mediated signals are mainly pro-inflammatory. b | Shielding of the β‑glucans by
mannans in hyphae prevents activation of the dectin 1 signalling pathways. c | Inhibitory signals from complement
receptor 3 (CR3) and Fcγ receptor II (FcγRII)/FcγRIII have inhibitory effects on the activation of the immune system via
TLR4. ERK, extracellular signal related kinase; MyD88, myeloid differentiation primary response gene 88; NF‑κB, nuclear
factor κB; Syk, spleen tyrosine kinase, TLR, Toll-like receptor.

another study in dectin 1–/– mice, which failed to discern
increased susceptibility to candidiasis106. Differences in
the experimental host or between different C. albicans
strains might account for these discrepancies, and addi-
tional studies are needed to pinpoint the origin of these
differences.
Studies on the in vivo roles of other LRs in the
response to C. albicans defences are also scarce. In one
study, intraperitoneal administration of C. albicans
resulted in an increased fungal burden in the organs
of MR–/– mice, compared to controls, although survival
was not affected107. However, intraperitoneal injection
of C. albicans is not an established experimental model
of invasive candidiasis, as it mainly induces peritonitis
rather than a disseminated infection108. A recent study
has demonstrated an important role for galectin 3 in
the recognition of C. albicans in the gut29, with β-(1,2)-
mannosides able to block colonization and invasion of
C. albicans in the intestines9. Finally, like mice defective
in TLR2, mice defective in CR3 or FcγR are more resistant
to disseminated candidiasis, and display more vigorous
cytokine release and antifungal killing, in line with the
immunosuppressive effect of these receptor pathways on
the inflammatory response60.
It has become clear that PRRs are important for the
host response to C. albicans, with various TLRs and LRs
having distinctive roles in innate immunity: some recep-
tors have a more pro-inflammatory role (for example,
TLR4, dectin 1 and the MR), whereas others exert immu-
nosuppressive effects (for example, TLR2, CR3 and FcγR)
(FIGS 3,4). In addition, the choice of experimental model
and the C. albicans strain used might have an important
impact on the outcome of infection. More work must
still be done to fully understand the role of PRRs in the
various types of C. albicans infections in vivo. Studies of
disseminated candidiasis are more advanced, but little
work has been done in other forms of C. albicans infec-
tion, such as oropharyngeal or vaginal candidiasis, and
little attention has been paid to the mechanisms that lead
to colonization rather than infection.
Escape mechanisms based on PRRs
As evidence supporting the important role of PRRs
in the recognition of C. albicans for the induction
of an immune response accumulates, data have also
emerged to show that certain fungal pathogens have
evolved strategies to curtail recognition by these recep-
tors, or to use it to their own advantage to evade the
host response. For example, it was recently shown in
Drosophila melanogaster that Gram-negative bind-
ing protein 3 (GNBP3) acts as a dectin-1-like lectin-
recognition mechanism for fungal glucans, including
that of C. albicans109. Certain fungal insect pathogens
secrete proteases that degrade GNBP3 to avoid detec-
tion, but the D. melanogaster persophone protease is
activated by the fungal protease virulence factor, which
results in the bypass of GNBP3 and the activation of the
Toll pathway downstream of the PRR. It remains to be
seen whether there is a resonance of this co-evolution
between recognition mechanisms and virulence factors
in human–fungal interactions.
In the mammalian host, inappropriate or premature
activation of immunomodulatory receptors can be used
as an escape route by C. albicans, as detailed above for
the outcome of systemic candidiasis in TLR2–/– mice
(FIG. 4a). Modulation of DC function can also be a target

74 | january 2008 | volume 6 www.nature.com/reviews/micro

© 2008 Nature Publishing Group

for escape mechanisms, with the MR and DC‑SIGN pos-
sibly inducing anti-inflammatory cytokine production
and immunosuppression110,111. Thus it seems that the
MR might have a dual role, either pro-inflammatory (in
monocytes and macrophages) or immunosuppressive
(in DCs), although this assumption is still controver-
sial. In addition, the recognition of β‑glucans by CR3
does not trigger protective host responses, such as the
respiratory burst42, and can repress pro-inflammatory
signals44 (FIG. 4). In line with these immunosuppressive
effects in vitro, CR3-knockout mice are more resistant
to disseminated candidiasis60, and are also more resist-
ant to Blastomyces dermatitidis infection43. Some studies
have suggested that the differential activation of stimula-
tory and inhibitory receptors is caused by the dimorphic
nature of C. albicans yeast and hyphae, which induce dif-
ferent DC activation profiles. The recognition of yeasts
by DCs is mediated mainly by the MR, dectin 1 (Ref. 60)
and DC‑SIGN27,112, and hyphae are recognized mainly
through dectin 2, CR3, FcγRII and FcγRIII60.
In addition to inducing direct anti-inflamma-
tory effects, C. albicans has developed strategies to
either block or avoid recognition by stimulatory PRRs
(FIG. 4b) . The morphogenetic change of C. albicans
from a yeast to a hyphal form, after adhesion of yeast
cells to the intravascular endothelium and invasion of
the surrounding tissue, might represent a mechanism
whereby the pathogen can avoid recognition of β‑glucans
by the immune system113. Hyphal filaments lack surface-
exposed β‑glucan and have a range of other cell-surface
modifications that could influence immune detection11.
In addition, the induction of the pro-inflammatory
response through dectin 1 can be prevented, because the
mannoprotein layer covers the β‑glucan layer of the cell
wall, thus blocking the β‑glucan–dectin 1 interaction11.
Accordingly, exposure of β‑glucan through treatment
with the antifungal caspofungin, by heat treatment of
cells or by mutation of glycosylation genes that result
in depletion of surface mannan all led to an enhanced
dectin-1-mediated pro-inflammatory cytokine signal.
This suggests that the mannoprotein layer is a mask that
downregulates the pro-inflammatory dectin-1-mediated
response114. As the exposed β‑glucan of intact C. albicans
yeast cells is thought to be presented mainly at the sur-
face of the wall as bud scars, the attenuation of dectin‑1-
mediated recognition during germ tube evagination
could be due to the expansion of the hyphal cell surface
that lacks any bud scars. In line with this, Rappleye et al.
showed that the β‑glucan–dectin 1 interaction between
Histoplasma capsulatum and macrophages can be
shielded by a different cell wall component — α-(1,3)-
glucan115. The presence of α-(1,3)-glucan in yeast cells
prevents recognition through dectin 1, and mutation of
AGS1 (which encodes α-(1,3)-glucan synthase) results
in greatly enhanced TNFα production115. Therefore,
both mannans and α‑glycans can act as shields that mask
dectin-1-mediated immune responses.
It is important to note that mannans are in them-
selves immunostimulatory 15,24,116–118. How can the
immuno­stimulatory properties of mannans be recon-
ciled with the apparent inhibitory effect of masking
nature reviews | microbiology volume 6 | january 2008 | 75
© 2008 Nature Publishing Group
REVIEWS
the β-glucan–dectin 1 interactions? The answer to this
paradox might reside in the complex interaction between
TLRs and LRs. Although both mannans and glucans can
induce pro-inflammatory signals, concomitant stimula-
tion of the mannan–TLR and β-glucan–dectin 1 path-
ways has a synergistic effect on the amplification of the
immune response. Amplification mechanisms between
dectin 1 and TLR2 (Refs 49,50), and between dectin 1
and TLR4 (K. M. Dennehy and colleagues, unpublished
observations) have recently been documented. If the
interaction of β-glucans with dectin 1 is shielded by
mannans, this synergism may be lost, with inefficient
activation of host defence, despite recognition of man-
nans. It is therefore mainly the loss of synergistic effects,
rather than the inhibition of an individual recognition
pathway, that may lead to defective cytokine release.
An integrated model of innate recognition
Progress in our understanding of the mechanisms respon-
sible for C. albicans recognition, as described above,
allows us to suggest an integrated view of how C. albicans
is recognized and of how the host innate immune
response is activated during candidiasis. Although we
are still in the early stages of understanding the com-
plexity of fungal recognition, and there is still much
to learn, there are several principles that characterize
recognition of C. albicans.
• First, recognition depends on ‘tasting’ several
PAMPs in the fungal cell wall. Specific receptor sys-
tems have evolved for the recognition of the major
polysaccharide cell wall components, such as the
MR and DC‑SIGN for the recognition of branched
N‑linked mannan, TLR4 for linear O‑linked mannan,
galectin 3 for β-mannosides, dectin 1 and TLR2 for
β-glucan and PLM, and CR3 for β-(1,6)-glucan.
• Second, despite overlapping and sometimes redun-
dant functions, each ligand–receptor system activates
specific intracellular signalling pathways, and this has
distinct consequences for the activation of the various
components of the host immune response.
• Third, differential expression of the various PRRs is
an important mechanism for the cell-type-specific
response to fungal pathogens (FIG. 2).
• Fourth, the fully integrated response to a specific
pathogen depends on the mosaic of PRRs and recep-
tor complexes that are engaged. Co-stimulation via
multiple PAMP–PRR interactions can increase both
the sensitivity and the specificity of the immune rec-
ognition process.
These four principles can be extrapolated to immune
recognition of any microorganism. For example, the
final response upon stimulation of monocytes by
C. albicans depends on integrating the signals that are
received from TLR2, dectin 1, TLR4 and the MR. This
response differs from the stimulation that is induced
by the Gram­-negative bacterium Escherichia coli, which
can be ascribed to recognition of lipopolysaccharide by
TLR4, membrane lipopeptides by TLR2 and flagellin by
TLR5. By contrast, Gram-positive bacteria are mainly rec-
ognized by TLR2 (which recognizes lipoteichoic acid) and
NOD2 (which recognizes peptidoglycans). The different
REVIEWS

intracellular events that are stimulated by these pathways
are ultimately responsible for the tailored innate response
to infection by different classes of microorganisms.
From this perspective, although described here for
C. albicans, these general principles can be considered
as a blueprint for pattern recognition of all pathogenic
microorganisms by the innate immune response.
Future directions
In parallel with the general renaissance in the field of
innate immunity, our understanding of C. albicans rec-
ognition by the innate immune system has improved
considerably over the past decade. Despite this progress,
more challenges lie ahead. Although we have gained a
much better understanding of the basal (polysaccharide)
structures that are recognized by the PRRs, more work
must be done to understand the details of the interac-
tions of these PAMPs with their receptors on the host
cell membrane. Recent evidence, for example, demon-
strates that dectin 1 associates with tetraspanins, which
suggests that dectin 1 forms receptor complexes on the
cell surface that might be involved in regulating immune
responses88. In addition, little is known about the recog-
nition of the other components of the C. albicans cell
wall, such as the proteins themselves, or the possible
sensing of products that are secreted by C. albicans.
The differential recognition of the yeast and hyphal
forms also promises to be a fertile area for future
research.
An additional challenge is to provide evidence that the
pattern-recognition pathways identified in experimental
models are also applicable during C. albicans infections in
humans. This challenge can be partly met by experiments
in which human primary cells are exposed to C. albicans.
However, valuable information can also be gathered from
immunogenetic studies. For example, TLR4 polymor-
phisms have recently been identified as a susceptibility
trait for systemic candidiasis, which supports a role for
this receptor in the host immune response to C. albicans119.
Studies of additional functional polymorphisms in larger
cohorts of patients are needed to confirm the role of the
various pathways in the pathogenesis of candidiasis
The ultimate ambition of research into the biology of
host–fungus interactions is to be able to translate findings at
the molecular level of the interactions into new treatments
for patients who are vulnerable to lethal fungal infections.
Although direct blockade or stimulation of PRRs might
be problematic as a viable therapeutic approach, vaccine
biology has become a domain in which our newly acquired
knowledge of PRRs is already demonstrating potential120.
The understanding of the modulation of DC function by
PRRs has led to the design of novel and specific vaccine
adjuvants, and this work is likely to have a major impact on
the development of future antifungal vaccines.
In conclusion, in this Review we have presented
an integrated model of innate pattern recognition
of an important human pathogen, the fungus C. albicans,
including a description of the PAMPs of C. albicans that
are recognized by the host, to the receptor pathways
that are stimulated by specific fungal molecular struc-
tures. We have also discussed and speculated on how these
signals are integrated to bring about efficient activation
of the host innate response. This model, which focuses
on the interaction of C. albicans with host defences,
could be conceptually pertinent to the modelling
of various other host–pathogen interactions.

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Acknowledgements
This work was supported by a Vidi Grant from Netherlands
Organization for Scientific Research to M.G.N., and by the
Wellcome Trust to N.A.R.G. and G.D.B.
DATABASES
Entrez Genome project: http://www.ncbi.nlm.nih.gov/sites/
entrez?db=genomeprj
Borrelia burgdorferi | Candida albicans |
Saccharomyces cerevisiae | Schistosoma mansoni
UniProtKB: http://ca.expasy.org/sprot
CARD9 | dectin 1 | dectin 2 | galectin 3 | IL-2 | IL‑6 | IL‑10 | Syk |
TLR2 | TLR4 | TLR6 | TLR9
All links are active in the online pdf
 REVIEWS

Kiss and spit: the dual roles of

Toxoplasma rhoptries
John C. Boothroyd* and Jean-Francois Dubremetz‡
Abstract | Toxoplasma gondii is a single-celled, eukaryotic parasite that can only
reproduce inside a host cell. Upon entry, this Apicomplexan parasite co-opts host
functions for its own purposes. An unusual set of apical organelles, named rhoptries,
contain some of the machinery that is used by T. gondii both for invasion and to
commandeer host functions. Of particular interest are a group of injected protein
kinases that are among the most variable of all the T. gondii proteins. At least one of
these kinases has a major effect on host-gene expression, including the modulation
of key regulators of the immune response. Here, we discuss these recent findings and
use them to propose a model in which an expansion of host range is a major force that
drives rhoptry-protein evolution.

Apicomplexa
A phylum of unicellular
eukaryotes that are obligate
parasites and defined by a
collection of apical organelles
that are involved in invasion of
a host cell.
Microneme
A small, cylindrical organelle
that is found at the periphery
of the anterior end of
Apicomplexan parasites that
secretes its contents onto the
surface of a gliding or invading
parasite.
*Department of Microbiology
and Immunology, Stanford
University School of Medicine,
Stanford, California
94305‑5124, USA.
‡
UMR 5539 CNRS, Université
de Montpellier 2, CP 107,
Place Eugène Bataillon,
Montpellier 34090, France.
Correspondence to J.C.B.
e‑mail: john.boothroyd@
stanford.edu
doi:10.1038/nrmicro1800
Published online
3 December 2007
The phylum Apicomplexa includes a large number of
obligate intracellular parasites. Among these are some
notorious human and animal pathogens from genera
such as Plasmodium (the causative agent of malaria),
Toxoplasma (an important cause of congenital disease
and infection in immuno-compromised patients1; see
BOX 1 for a brief description of the Toxoplasma life cycle
and the clinical spectrum of human toxoplasmosis),
Cryptosporidium (a cause of serious gastrointestinal
disease) and Eimeria (a major problem in the poultry
industry and cause of chicken coccidiosis). The phy-
lum is defined by the presence of an apical complex
that comprises a microtubule anchoring ring through
which dedicated secretory organelles release their con-
tents2. There are two types of apical secretory organelle
— the small, rod-shaped micronemes and the much
larger, bulb-shaped rhoptries (Greek for club). FIG. 1a
provides an electron micrograph of one of the asexual
forms of Toxoplasma gondii, which clearly shows an
apical cytoskeleton and a substantial complement of
micronemes and rhoptries.
Given their conservation across much of the phy-
lum, it has long been suspected that rhoptries have a
key role in the intracellular lifestyle of these pathogens.
Recently, exciting results have shed light on at least two
functions of the rhoptries. In this Review, we will discuss
the ultrastructure and content of rhoptries, their role in
invasion and function in subverting host-cell processes,
as well as the evolutionary pressures that might underlie
the extreme variability of rhoptry proteins.
Rhoptry ultrastructure and content
The size, electron density and number of rhoptries var-
ies among Apicomplexan species and between the dif-
ferent developmental stages of a single species. In this
Review, we will mainly focus on the rapidly dividing,
asexual form of T. gondii that is known as the tachyzoite3.
Tachyzoites predominate during the acute stages of
infection in an intermediate host, which can be virtually
any warm-blooded animal (BOX 1). This breadth of host
range also extends to the cellular level, as tachyzoites
can invade and replicate in almost any host cell that they
encounter, at least in vitro. In vivo, the cellular tropism
of tachyzoites has not been well characterized as, until
recently4,5, methods to survey parasite growth in the
entire animal have been lacking. Most in vitro studies use
fibroblasts as the host cell because of their availability,
the fact that they are a primary cell line (making studies
on host-gene expression more relevant than in a trans-
formed cell line) and the ease with which they can be
examined by light microscopy (as they have full contact
inhibition and grow as a uniform, flat monolayer).
Tachyzoites typically have ~12 rhoptries, each of
which is ~2 to 3 micrometres in length6 (FIG. 1). Using
light microscopy, the rhoptries can be visualized as an
elongated cluster that is present at the anterior end of
the parasite and is frequently located on one side. They
are visible using differential interference contrast micro-
scopy, but are more easily observed by immunofluores-
cence using a range of antibodies that are specific for their
protein contents. Interestingly, most of these antibodies

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REVIEWS
Box 1 | Toxoplasma gondii biology
Asexual Sexual
Warm-blooded intermediate Feline
4 termed ROP proteins) and 5 are present in the rhoptry
Bradyzoite Gamete
3 1 2 5 6
Tachyzoite Oocyst
7 that have yet to be verified as being truly rhoptry in
T. gondii is an extremely common protozoan parasite of warm-blooded animals that has
a host range that extends from birds to humans. Its life cycle comprises two, potentially
independent cycles, one asexual and one sexual, although Nature Reviewsbetween
movement | Microbiology
the two
is almost certainly key to efficient transmission (see the figure).
The asexual cycle (see the figure, left-hand panel) can occur in virtually any warm-
blooded animal and ~100 mammalian and avian species have been documented as
being infected with T. gondii in nature. In these ‘intermediate’ hosts, the parasite
population initially expands by rapid proliferation of the tachyzoite form (‘tachy’ means
fast in Greek and in this context refers to speedy replication). Once an immune response
is elicited, the parasite differentiates to the more slowly growing bradyzoite form
(‘brady’ means slow in Greek) (arrow 1). The bradyzoite form can persist for the life of the
host in cyst-like structures that are present deep in the brain and other tissues. During
immunosuppression (for example, in patients with AIDS) the parasite can resume rapid
replication in the form of tachyzoites (arrow 2). Transmission between intermediate
hosts is by the ingestion of raw or under-cooked meat and other organs that contain the
infectious, encysted bradyzoites (for example, a mouse eaten by a hawk or an under-
cooked lamb chop eaten by a human) (arrow 3). The sexual cycle (see the figure, right-
hand panel), which begins when bradyzoite-bearing tissue from an intermediate host is
ingested by a feline (arrow 4), involves gametogenesis and fertilization (arrow 5) in the
gut epithelium of felines. The cat family is the only group of animals that is known to
serve as a definitive host for this parasite; that is, it is the only host in which sexual
reproduction occurs, although felines can also support asexual reproduction. The
sexual cycle culminates in the shedding of up to 100,000,000 highly stable oocysts in the
faeces of an infected cat (these oocysts are initially shed in an immature state and
require approximately 2 days to mature in the environment and gain full infectivity).
Mature oocysts are highly infectious and can either infect another cat (arrow 6) or, more
probably, a grazing intermediate host (arrow 7) that is foraging in a farm.
T. gondii causes serious disease in humans, but this disease is primarily confined to
the developing foetus of a woman who acquires her first infection during pregnancy
and individuals who are immunocompromised as a result of HIV-1 infection,
lymphoma or other immune-suppressive syndromes. Disease pathologies range from
asymptomatic to severe and, sometimes, fatal55. Occasionally, otherwise healthy
adults can also experience acute symptoms, especially in the eye54. These different
disease outcomes may be related to which of the three most common strains of
T. gondii (Type I, II or III) is responsible for the infection46,47,55.
stain either the bulbous base7,8 or the tapering neck of the
Rhoptry rhoptry9, but not both. It seems, therefore, that the rhop-
A club-shaped secretory try contents are not a random mixture but are sorted
organelle that is found at the into discrete subcompartments. No internal membranes
anterior end of Apicomplexan
seem to separate these domains and the mechanism by
parasites that releases its
contents during invasion; which proteins are sorted to a particular region is not yet
subdivided into a bulbous known. After translation, at least initially, rhoptry pro-
base and tapering-neck. teins move through a conventional eukaryotic secretory
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© 2008 Nature Publishing Group
pathway that involves rough endoplasmic reticulum and
the Golgi apparatus, but the process of rhoptry forma-
tion and the signals that ultimately target proteins to this
organelle have yet to be precisely identified10–14. There is
some evidence that rhoptries are related to exosomes15,
which are membrane-limited bodies that are extruded
by some cells. This has interesting implications for the
final topology of a rhoptry protein during release, as
exosome formation might involve an invagination of
the rhoptry membrane and, therefore, place an integral
membrane protein in an inverted orientation relative to
their position by alternative models16.
There are 29 proven rhoptry proteins, of which 24 are
present in the rhoptry bulb (most of these are therefore
neck (termed RON proteins) (TABLE 1). Many of these
were first discovered by proteomic analyses of purified
rhoptries9, which identified an additional 28 proteins
origin. Given the high percentage of proteins that were
verified as being rhoptry proteins from the first group
that was analysed, most of these 28 proteins are prob-
ably also from this compartment. Further evidence of a
rhoptry origin comes from the fact that many of these 28
proteins are paralogues of known rhoptry proteins, as are
the predicted products of several additional genes that
have yet to be analysed in detail (TABLE 2).
The largest rhoptry gene family shows clear homol-
ogy to protein kinases17. The canonical member of this
family is ROP2 and, in at least one instance, a member
of this family (ROP18) has been confirmed to have
kinase activity17,18, although the vast majority seem to
have lost the key catalytic residues and, hence, the ability
to phosphorylate proteins19 (TABLE 1). Apart from their
kinase domains, these proteins seem to be unique to the
Toxoplasma genus and its close relatives (for example,
species of Neospora); no bona fide homologues have been
described in the distantly related Plasmodium spp. This
could reflect an incomplete knowledge of the rhoptry
proteome in species of Plasmodium and/or a difference
in the intracellular niches that they occupy. Other ROPs
are homologous to phosphatases20 and proteases21,22, but
several ROPs, including ROP1, ROP6 and ROP9, seem to
be unique to the Toxoplasma genus and are of unknown
function23,24.
In contrast to the extensive ROP2 family, each of the
RON proteins is encoded either by a unique gene that has
no similarity to any other gene in the T. gondii genome
(for example, RON1 and RON5) or by a gene that has
one or two paralogues elsewhere in the T. gondii genome
(for example, RON2, RON3 and RON4) (TABLE 2). The
initial discovery of the sequence of the RON proteins
gave no clue as to their biological function9. Several
T. gondii RONs have clear orthologues in related genera,
including Plasmodium, which suggested their involve-
ment in processes that are common to the Apicomplexa
phylum. Although their exact function has not been deter-
mined, the unusual secretion of RON4 and its subsequent
migration down the length of the parasite during invasion
has led to clear models about the overall role of RON4 and
other RONs, as discussed below.

Paralogue
A gene that shares a common
evolutionary origin and has
evolved in parallel with another
gene that is located in the
same genome or organism,
typically to serve different but
related functions.
Orthologue
A gene in one species that
shares a common evolutionary
origin with a related gene in a
different species and that
serves essentially the same
function.
Parasitophorous vacuole
The vacuole that harbours the
parasite.
Moving junction
(MJ). A migrating ring of
contact between a host-cell
plasma membrane and the
surface of an invading parasite.
nature reviews | microbiology volume 6 | january 2008 | 81
REVIEWS
Finally, in addition to their protein complement, a
rhoptries also contain lipids25 and this lipid comple-
ment has a high ratio of cholesterol to phospholipids. AC
Interestingly, these lipids are sometimes present as RON
membrane whorls inside the rhoptry organelle, which
can be visualized using electron microscopy. This might
explain the unusual topology of ROP proteins that are PVM
M
released into the host cell during invasion; as discussed
below, some ROPs seem to enter the host-cell cytosol in ROP
A
a freely soluble form whereas other ROPs are associated
with unusual vesicle-like bodies that seem to fuse with G
the nascent parasitophorous vacuole.
Role of rhoptries in cell invasion N
The only circumstance in which rhoptries are known to
secrete their contents is during the process of invasion
into a host cell (FIGS 2,3; see Supplementary information
S1 (movie)). The trigger for release is unknown, but it
evidently depends on a direct recognition between the
apical surface of the parasite and the receptor molecule
(or molecules) on the host cell. The identities of the mol-
ecules on either side of this interaction are also unknown
and no stimulus has yet been identified that will induce
rhoptry secretion in the absence of host-cell contact. The
mechanics of the fusion event that allows rhoptry pro-
tein release are a mystery. It could be as simple as fusion b
of the rhoptry to the parasite’s plasma membrane or,
perhaps, an intermediate, anterior compartment. Whatever
the process, a distinct opening at the anterior-most tip of PV
the tachyzoite is clearly observed during host-cell inva-
RON
sion26 and this is presumed to be the opening through HC
which the rhoptry contents ultimately flow. HPM
Once released, rhoptry proteins have various destina-
tions (FIG. 3). The RON proteins, RON2, 4 and 5, form PVM ROP
a complex with the micronemal protein AMA1 and
this multimeric complex colocalizes with the so-called
moving junction (MJ)27,28. The MJ is a ring-like structure
that represents the circular point of contact between the
parasite surface and host plasma membrane29. During
invasion, the MJ migrates down the length of the parasite Mito
(FIGS 2,3; see Supplementary information S1 (movie)).
Figure 1 | Toxoplasma gondii Nature
ultrastructure. a | An
Reviews | Microbiology
AMA1 seems to be necessary for the MJ to form, because intracellular T. gondii tachyzoite inside a parasitophorous
parasites in which AMA1 expression has been reduced vacuole membrane (PVM), showing the apical cytoskeleton
to ~1% of the wild type (using parasites that harbour a (AC) and neighbouring micronemes (M), rhoptry bulbs
tetracycline-regulated copy of the AMA1 gene) release (ROP) and rhoptry necks (RON). Other components of the
the RONs, but the MJ fails to form and the parasites do parasite, such as the nucleus (N), the Golgi apparatus (G)
not invade host cells28. and the plastid that is specific to the Apicomplexa phylum,
The MJ might represent the mechanism by which the ‘Apicoplast’ (A), are shown. The scale bar represents
the parasite makes contact with the host cytoskeleton. 0.5 µm. b | A lower magnification of a rosette of T. gondii in
the parasitophorous vacuole (PV) inside an infected cell
The most widely accepted model is one in which the
(HC). The host plasma membrane (HPM), as well as host
parasite anchors itself on integral proteins of the host
mitochondria (Mito) in close apposition to the PVM, can
plasma membrane and drags them backward (rela- also be seen. The scale bar represents 1 µm. Image in part a
tive to the parasite surface), effectively converting the reproduced, with permission, from REF. 56  Elsevier
host plasma membrane into parasitophorous vacuole Science. Image in part b reproduced, with permission, from
membrane that surrounds the intracellular portion of REF. 57  Elsevier Science.
the parasite (FIG. 2). This wrapping of the parasite in
parasitophorous vacuole membrane is simultaneous
with a clear forward motion of the parasite into the grip on something that is connected to fixed anchors
host cell, relative to the host-cell’s normal perim- within the host cell; that is, the host plasma membrane
eter (Supplementary information S1 (movie)), which integral proteins must be connected to the host-cell
strongly argues that the parasite must have a firm cytoskeleton. Finally, the model predicts that the
© 2008 Nature Publishing Group
REVIEWS

Table 1 | Known rhoptry proteins

Protein Gene identification Final Predicted Biological Comments Plasmodium Refs||
name number*; GenBank destination‡ coding function falciparum§
accession number function
ROP1 583.m00003; M71274 PV Unknown Unknown Knock-out in a type I strain is still No 23
virulent
ROP2A¶ 33.m01398; Z36906 PVM Protein kinase Mitochondria More adjacent genes probably exist No 37
recruitment but are missing from ToxoDB4.2
ROP2B¶ 63.m00146 PVM Protein kinase Unknown Should be a tandem, identical copy of No 37,58
ROP2A but appears as a fragment in
the ME49 sequence in ToxoDB4.2
ROP4¶ 83.m02145; Z71787 or PVM Non-catalytic Unknown Phosphorylated; mis-annotated in No 19,37
AY662677 kinase ToxoDB4.2 as one gene (fusion with
ROP7)
ROP5¶ 551.m00238; EF466101 PVM Non-catalytic Unknown Approximately 3 or 4 more tandem No 9,35,59
or DQ116423 kinase copies exist that are not present in
the ME49 sequence in ToxoDB4.2
ROP7¶ 83.m02145; AM056071 PVM Non-catalytic Unknown Mis-annotated in ToxoDB4.2 as one No 36
kinase gene (fusion with ROP4)
ROP8¶ 33.m00005; AF011377 PVM Non-catalytic Unknown None No 58
kinase
ROP9 49.m00048; AJ401616 Unknown Unknown Unknown Distinct from another protein No 24
named ROP9 (Ref. 60), for which the
sequence and gene are unidentified
ROP10 583.m05686; DQ124368 Unknown Unknown Unknown None No 9
ROP11¶ 42.m03584; AAZ29607 Unknown Protein kinase Unknown None No 9
or DQ077905
ROP12 20.m08222; DQ096559 Unknown Unknown Unknown None No 9
ROP13 583.m09115; DQ096560 Unknown Unknown Unknown Mis-annotated in ToxoDB4.2; No 9
encoded on the opposite strand
ROP14 583.m00692; DQ096565 Unknown Transmembrane Unknown None Yes 9
ROP15 27.m00091; DQ096561 Unknown Unknown Unknown None No 9
ROP16¶ 55.m08219; DQ116422 Host nucleus Protein kinase Host subversion Extremely different in T. gondii type I, No 9
and virulence II and III strains
ROP17¶ 55.m08191; AM075203 Unknown Protein kinase Unknown None No 17
ROP18¶ 20.m03896; AM075204 PVM Protein kinase Virulence Extremely different in T. gondii type I, No 17
or EF092842 II and III strains
TgSUB2 583.m00011; AF420596 Unknown Serine protease ROP protein None Yes 22
processing
Toxopain1 50.m00008; AY071839 Unknown Cathepsin B ROP protein None No 21
protease processing
TgPP2C-hn 74.m00766 + 74m.00767; Host nucleus Protein Unknown Injected into host cell where it ends No 20
EF450457 phosphatase up in the nucleus; knock-out has a
2C slight growth defect in vitro
TgNHE2 129.m00252; AY735393 Unknown Na+ and H+ Ionic Knock-out in type I strain is still Yes 61
exchanger homeostasis virulent
Toxofilin 33.m02185; AJ132777 Unknown Actin-binding Unknown Possible interference with host actin No 62
BRP1 583.m09133 Unknown Unknown Bradyzoite- and Knock-out in type II strain is still No 63
merozoite- virulent
specific
TgRAB11 80.m00009 Unknown Small GTPase Protein None Yes 9
trafficking
RON1 583.m11443 + 583. Unknown Possibly GPI- Unknown None Yes 9
m00597; DQ096562 anchored
RON2 145.m00331; DQ096563 MJ Transmembrane Invasion Covalently attached to RON4 Yes 9
RON3 42.m00026; DQ096564 Unknown Transmembrane Unknown None Yes 9
RON4 44.m06355; DQ096566 MJ Unknown Invasion Covalently attached to RON2 Yes 9
RON5# 583.m09191 + 583. Possibly MJ Unknown Invasion Cleaved into three major fragments Yes None
m09192 + 583.m00636
*The ToxoDB4.2 (Toxoplasma gondii Genome resource; see Further information64) gene identifier is provided for the T. gondii ME49 strain, along with the GenBank
entry for (typically, but not always) the T. gondii RH strain, if such an entry exists. The GenBank entry should be a verified coding sequence and, therefore, encode the
definitive amino-acid sequence of the primary translation product. The gene identifiers in ToxoDB4.2 are tentative predictions that in most cases have yet to be
experimentally confirmed. Consequently, although they are approximately correct, the transcription start sites, splice junctions and predicted protein sequences
might be incorrect. This is why some genes have multiple identifiers — for example, the large RON1 gene is currently incorrectly annotated as two different genes
(indicated by the + symbol, which separates the gene identifiers listed). ‡ Final location of the protein after invasion. § Significant orthologue, with homology that
extends beyond the presence of a conserved enzymatic domain (for example, more than just a kinase or protease active site is conserved throughout evolution),
exists in P. falciparum. ||The reference that reports the definitive sequence and/or identification of this protein as a rhoptry protein. ¶Homologue of ROP2. No gene has
yet been linked to the ROP2 family member that has been dubbed ROP3. ROP3 could simply be a post-translational modification of one of the known ROP2 family
members or encoded by one of the ROP2-like genes for which the protein product has yet to be detected. #P. Bradley and M. Lebrun, unpublished observations.
GPI, glycophosphatidylinositol; MJ, moving junction; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane.

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Table 2 | Suspected rhoptry proteins

Protein Gene identification Final Predicted coding Comments Plasmodium Refs||
name number*; GenBank destination‡ function falciparum§
accession number
ROP6 55.m00092; AY792971 Possibly HPM Possible protease and None No 665
GPI-anchored
ROP2L3¶ 55.m08224 Unknown Protein kinase Mass spectrometry data for expression**; No 9,17
detected in rhoptry proteome‡‡
ROP2L4¶ 57.m01774 Unknown Protein kinase None No 17
ROP2L5¶ 49.m03275 Unknown Protein kinase None No 17
ROP2L6 ¶
80.m02343 Unknown Non-catalytic kinase Mass spectrometry data for expression**; No 9,17
detected in rhoptry proteome‡‡
ROP2L7¶ 49.m03159 Unknown Protein kinase None No None
ROP2L8 ¶
52.m01543 Unknown Protein kinase Mass spectrometry data for expression**; No 9
detected in rhoptry proteome‡‡
ROP2L9¶ 55.m04788 Unknown Protein kinase Mass spectrometry data for expression** No None
ROP2L10¶ 59.m06126 Unknown Protein kinase None No None
ROP2L11 ¶
86.m00398 Unknown Unknown Truncated pseudogene No None
ROP2L12¶ 86.m00844 Unknown Unknown Truncated pseudogene No None
ROP2L13 ¶
25.m01746 Unknown Non-catalytic kinase None No None
RON2L1# 83.m01266 Unknown Unknown None Yes 28
RON2L2# 57.m01722 Unknown Unknown None Yes 28
RON3L1 #
20.m03905 Unknown Unknown Mass spectrometry data for expression** Yes None
RON4L1# 52.m01582 Unknown Unknown Mass spectrometry data for expression** Yes 28
*The ToxoDB4.2 (Toxoplasma gondii Genome resource; see Further information64) gene identifier is provided for the T. gondii ME49 strain, along with the GenBank
entry for (typically, but not always) the T. gondii RH strain, if such an entry exists. The GenBank entry should be a verified coding sequence and, therefore, encode
the definitive amino-acid sequence of the primary translation product. The gene identifiers in ToxoDB4.2 are tentative predictions that in most cases have yet to be
experimentally confirmed. Consequently, although they are approximately correct, the transcription start sites, splice junctions and predicted protein sequences
might be incorrect in their detail. ‡Final location of the protein after invasion. § Significant orthologue, with homology that extends beyond the presence of a
conserved enzymatic domain (for example, more than just a kinase or protease active site is conserved throughout evolution), exists in P. falciparum. ||Reference
that originally reported this gene or protein. ¶ROP2-like (ROP2L) protein predicted in ToxoDB4.2 but not yet confirmed to be rhoptry localized and, except as
noted, not even confirmed to be expressed. #RON2-, RON3- or RON4-like protein (RON2L, RON3L and RON4L, respectively) predicted in ToxoDB4.2 but not yet
confirmed to be rhoptry localized and, except as noted, not even confirmed to be expressed. **Mass spectrometry has revealed that one or more peptides are
present in tachyzoites based on data presented in ToxoDB4.2. ‡‡Detected by mass spectrometry in rhoptry-enriched fraction but not yet confirmed to be rhoptry-
localized. The biological function of these proteins is unknown. GPI, glycophosphatidylinositol; HPM, host plasma membrane.

region of contact between the parasite and the host
cell needs to be a circular band, as otherwise a gliding
parasite would not be able to take a ‘dive’ into the host
cell — it would simply keep moving along the sur-
face. AMA1, which was first described in Plasmodium
falciparum, might organize the MJ into a ring28. The
association of AMA1 with RON4 is also observed in
P. falciparum, which indicates that the overall collabo-
ration of rhoptry RONs with micronemal AMA1 is
a conserved feature of most, if not all, species of the
Apicomplexa phylum30.
How the RON proteins (RON2, 4 and 5) that
form the MJ complex associate with each other is
not known, although RON2 and RON4 seem to be
linked by disulphide bonds 28. How these proteins
further associate with a micronemal protein such as
AMA1 is also unclear; it is presumed, however, that
they come into contact with one another at, or just
below, the apical surface of the parasite. Likewise,
the topology of the various components of the MJ
complex within the membrane (or membranes) has
not yet been determined. RON2 has at least 2, and
possibly 3, predicted transmembrane domains, but it
is unclear which membrane (host or parasite) they
are embedded within. One exciting possibility is that
RON2, or a different MJ protein, is inserted into the
host plasma membrane during the first steps of inva-
sion and that it then contacts the host cytoskeleton to
provide the anchoring that is described above. This is
analogous to a phenomenon that has been reported
for enteropathogenic Escherichia coli, in which the
bacterium inserts a protein (Tir) into the host cell
that then plays a part in attachment and subsequent
invasion31. An appealing feature of such a model is
that it would explain the ability of T. gondii to invade
almost any cell from a wide range of warm-blooded
animals; if they provide their own anchor, they could
make a home in almost any port, as long as they
are able to make contact with a highly conserved
cytoskeletal protein. Alternatively, of course, the
MJ proteins could remain anchored in the parasite’s
plasma membrane and associate with host-plasma-
membrane structures that are, in turn, anchored to
the host-cell cytoskeleton. If so, a portion, or domain,
of that host‑plasma‑membrane molecule would need
to be structurally conserved among many species and
cell types to explain the extraordinary range of hosts
and cells that T. gondii can productively infect.

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REVIEWS
HC
AC
* are observed in a small, but significant, percentage of
MJ
PVM
MJ
Figure 2 | Toxoplasma gondii invasion. A T. gondii
Nature Reviews | Microbiology
tachyzoite invading an HeLa cell (HC). An irregularly
shaped organelle that is derived from rhoptry exocytosis
(asterisk) is found near the apical cytoskeleton (AC). The
parasitophorous vacuole membrane (PVM) that is derived
from the host cell membrane is found around the portion
of the parasite that has invaded the host cell. The invasion
is thought to be driven by parasite motors acting at the
moving junction (MJ). The scale bar represents 0.5 µm.
Image reproduced, with permission, from REF. 56 
Elsevier Science.
ROP proteins are injected into host cells
ROPs seem to have completely different functions
from RONs. Like the neck proteins, ROPs are released
during invasion but they do not form organized struc-
tures and are not found at the MJ. Instead, following
release, they migrate to one of three general locations:
the lumen of the nascent parasitophorous vacuole; the
parasitophorous vacuole membrane; or the interior of
the host cell (FIG. 3).
ROP1 is an example of migration to the first loca-
tion: it is released during invasion and accumulates
within the lumen of the nascent parasitophorous
vacuole23. Remarkably, based on studies that used a
combination of ROP1 knock-out parasites and parasite
lines that express epitope-tagged versions of ROP1, it
seems that ROP1 can be synthesized in one parasite
but end up in the parasitophorous vacuole of another32.
This suggests that ROP1 is not simply ‘dumped’ into
the nascent parasitophorous vacuole during inva-
sion, but instead is released into the host cell where it
then migrates to the nearest parasitophorous vacuole.
Because in nature most cells will be infected by only
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© 2008 Nature Publishing Group
one parasite, the nearest parasitophorous vacuole is
usually that of the same parasite that released the ROP1
during invasion. Small vesicle-like structures referred
to as evacuoles have been observed that contain ROP1
but are devoid of parasites (hence they have also been
named ‘empty’-vacuoles or ‘e’-vacuoles32). Evacuoles
invasion events in tissue culture and their frequency
can be increased if invasion is blocked by drug treat-
ment. For example, treatment with cytochalasin D
prevents the actin and myosin motors of the parasite
from exerting their propulsive force, although attach-
ment is not affected (the host cell’s actin and myosin
motors appear to be irrelevant to parasite invasion17,33).
The result is a ‘frustrated’ parasite that is stuck to the
outside of a host cell that contains many evacuoles but
lacks a developing parasitophorous vacuole membrane
for the ROP1-containing evacuoles to fuse to.
The ROP2 family of proteins generally migrates to
the second location for rhoptry proteins, the parasito-
phorous vacuole membrane19,34–37. It seems that several
members of this protein family are intimately associated
with the parasitophorous vacuole membrane, and are
possibly even integral membrane proteins. Early sug-
gestions that a hydrophobic alpha helix functions as a
transmembrane domain34 have been called into ques-
tion now that we know that this helix is a conserved
feature of most protein kinases and its hydrophobicity
is a necessary feature of its being buried within the
interior of the protein. Although no crystal structure
of a ROP2 family member has been reported, it seems
unlikely that a helix would be used to span a membrane,
especially given the clear conservation of sequence (and
presumably structure) on either side of the hydrophobic
portion. ROP2 has also been implicated in the recruit-
ment of host-cell mitochondria 38,39. This has been
postulated to be through recognition of the processed
amino terminus of ROP2, which resembles a mitochon-
drial-import signal (that is, an amphipathic helix). The
proposal is that host mitochondria mistakenly attempt
to import ROP2, which is somehow firmly tethered to
the parasitophorous vacuole membrane. The result is
that as the mitochondria attempt to ‘reel in’ the ROP2
protein, they ratchet down onto the parasitophorous
vacuole membrane, which is where they are routinely
observed by electron microscopy.
Recent data on ROP18, a member of the ROP2
family, might indicate an interesting aspect of its asso-
ciation with the parasitophorous vacuole membrane;
when expressed inside an infected host cell, by direct
transfection of the ROP18 gene (minus the portion
encoding a signal peptide), the protein is eventually
found concentrated on the parasitophorous vacuole
membrane, presumably on the face that is exposed to
the host cytosol18. This is also the location of a putative
parasite-derived kinase that phosphorylates host iκB40.
Whether ROP18 or another member of the ROP2 fam-
ily is responsible for iκB phosphorylation remains to be
directly investigated and the mechanism by which these
proteins associate with the parasitophorous vacuole
membrane is likewise unknown.

Nucleus
Rhoptry bulb
Moving
Microneme junction Host plasma
Rhoptry neck membrane
Toxofilin?
PVM
ROP2 family
ROP1
ROP16
PP2C-hn
Nucleus
Figure 3 | Schematic model for rhoptry contribution to invasion. Rhoptry bulbs
(grey) and rhoptry necks (red) release their contents during the invasion
Nature process
Reviews in
| Microbiology
concert with simultaneous release by micronemes (green). RON2, RON4 and RON5
collaborate with micronemal AMA1 to create the moving junction, which migrates
down the surface of the parasite, forming a ring of contact with the host plasma
membrane. This effectively excludes many host plasma membrane integral proteins and
results in the generation of a parasitophorous vacuole membrane (PVM), which
envelopes the parasite. ROP2 family members are injected during invasion, perhaps in
association with small vesicles, and ultimately end up on the host cytosolic side of the
PVM. ROP1 is also observed in association with the injected vesicles, but most of this
protein ends up inside the parasitophorous vacuole lumen. ROP16 and PP2C-hn (hn is
the abbreviation for host nucleus) are not observed in the vesicles, but accumulate inside
the host nucleus. Other soluble rhoptry proteins (for example, toxofilin) are also
presumed to be injected, but in the absence of a concentrating mechanism they will be
present at too low a concentration to be detectable (only a few rhoptries secrete their
contents during invasion and any proteins that they release will be diluted by up to a
million-fold or more in the host cytosol). Figure adapted from Ref. 28.
The third known destination for ROPs is the interior
of the host cell or, more specifically, the host-cell nucleus.
So far, two rhoptry proteins have been observed in
the nucleus, a protein phosphatase of the 2C class
(PP2C-hn; hn is the abbreviation for host nucleus)20
and a putative protein kinase that is a highly diver-
gent member of the ROP2 family (ROP16 (Ref. 41)).
As for the other ROPs, how these proteins enter
the host cell is a mystery. The only clues come from
patch-clamp experiments, which show that there is a
break in the continuity of the host plasma membrane
at the earliest times of invasion42, perhaps reflecting
the moment when injection occurs (FIGS 2,3). The proc-
ess is clearly an early one, as both ROP16 and PP2C-hn
are detectable in the host nucleus within 15 minutes of
allowing infection to commence, which is close to the
time that such proteins would need to reach the host
nucleus if they were introduced at time zero. It should
be noted that once ROP16 and PP2C-hn enter the
host cytosol, conventional nuclear localization signals
(NLSs) are used to traffic them to the nucleus. This has
been demonstrated by showing that ablation of a classic
NLS signature, which both proteins have, stops these
nature reviews | microbiology volume 6 | january 2008 | 85
© 2008 Nature Publishing Group
REVIEWS
proteins from concentrating in the nucleus41,43. There
is no evidence for the existence in T. gondii of genes or
proteins that are related to those used by bacteria for
introducing proteins into a host cell (for example, type
III or type IV secretion systems44), although there are
many parallels between these processes.
The function of the rhoptry PP2C-hn is unknown,
as a knock-out strain showed no changes in host-gene
expression (based on microarray analysis of infected
cells) or virulence (based on infection studies in mice),
although the knock-out strain was partially compro-
mised in its ability to grow in fibroblasts in vitro43. Any
conclusion concerning the lack of a virulence phenotype
must be qualified, however, because the knock-out of
PP2C-hn was in an RH strain in which virulence is so
high (LD100 of 1 parasite) that anything short of a dra-
matic reduction in virulence might still yield a parasite
that is capable of killing a mouse.
ROP16 and ROP18 — roles in virulence
It has long been known that different strains of T. gondii
produce radically different pathologies in mice and
maybe even in humans45. Using F1 progeny from crosses
between two strains that differ in their virulence, two
research groups have mapped the parasite loci that are
responsible for the different pathogenicities46,47. The
results showed that virulence in a given host (in these
cases, mice) is influenced by which allele is present at
each of at least five loci. Two of the most important
loci are ROP16 and ROP18; for example, depending on
which allele of ROP18 a strain carries, its LD50 in mice
can vary by over 4 logs. The impact of the ROP16 locus
is less dramatic but still significant. The identities of the
other three virulence loci have yet to be determined.
For ROP18, there is still much to be learned about
the exact mechanism by which the different alleles effect
such dramatic differences in disease outcome. It is known
that the allele that is associated with low virulence yields
a tiny fraction (~0.1%) of ROP18 mRNA compared
with the amount that is produced by the alleles found
in more virulent strains46,47. This seems to be due to the
presence of a large insertion and a small deletion within
the presumptive promoter region of the low virulence
allele, which seems to render the promoter inactive. The
precise function of the ROP18 protein, however, is not
yet known, although it clearly does have potent kinase
activity, and a parasite substrate has been observed but
not yet identified18.
ROP16 subverts host gene expression
Microarray experiments using infected host cells in vitro
have shown that the infected host cell responds differ-
ently depending on which strain of T. gondii is used and
that these differences also segregate as distinct pheno-
types among the F1 progeny41. Importantly, ROP16 is
among the main parasite loci that have been shown to
be responsible for these differences. Testing of specifi-
cally engineered strains showed that this putative protein
kinase somehow intersects the host signal transducer
and activator of transcription (STAT) pathways41. These
pathways are central to the regulation of many host genes,
REVIEWS
including several cytokines and other immune-response
mediators. This suggested the obvious possibility that
differences in how a host cell responds to T. gondii might
result in differences in the disease pathology. One of
the main immune mediators that ROP16 affects by the
perturbation of STAT pathways seems to be interleukin
(IL)-12, which is central to the host response to T. gondii
infection48,49. Too much, or too little, IL-12 can have seri-
ous and negative consequences for the host by causing
too much or too little of the necessary immune responses,
and so its variable expression, which depends on the
allele of ROP16 that is carried by the invading parasite, is
an attractive explanation for the differences in the disease
that are caused by the various strains. This possibility
has been explored in vivo and ROP16 does indeed seem
to have a role in determining serum IL-12 levels and,
therefore, the overall course of infection41.
Using the terminology that is favoured by those
studying bacterial pathogens, ROPs are crucial ‘effec-
tor proteins’. They might equally be called ‘negotiator
proteins’, owing to their role in managing the interac-
tion between host and parasite, as the vast majority of
T. gondii infections result in a persistent infection with
little, if any, disease.
Rhoptries and T. gondii evolution
ROP16 and ROP18 are members of a large gene family
(the ROP2 family) and are two of the most variable
loci in the entire T. gondii genome. The ratio of syn-
onymous to nonsynonymous substitutions is also
extremely high, thus strengthening the argument
that these two genes are subject to a strong positive
selection for change41,46,47. What is the pressure that
drives this process? One possibility is simply that it is
the immune pressure of the sort that drives variation
in many pathogen proteins. ROP16 and ROP18 may
particularly be subject to such selection, relative to
other T. gondii antigens, because they seem to be
accessible to the host cytosol and are, therefore, freely
available to the host cell for class I major histocompat-
ibility complex presentation. As yet, however, there are
no data to indicate that these proteins are efficiently
presented or are crucial as targets of the immune
response in the conventional sense. This is in contrast
to the well-characterized family of surface antigens
that is known as the SAG1 related sequences or SRS
family, which are immunodominant antigens (at least
for the humoral response) that show a more modest
level of variability between strains50.
An alternative hypothesis for the diversification of
ROP16 and ROP18 is selection for an expanded host
range. This model posits that if a strain found itself in
a new ecological niche, and therefore in a new host, as
long as it could infect to some degree (and be trans-
mitted), there would be a strong pressure to optimize
the ‘fit’ between a negotiator protein and the new
host. This could lead to a powerful and rapid selec-
tion for new gene variants or, as is the case for ROP18,
selection for dramatic events that downregulate or
upregulate its expression (as discussed above, the
ROP18 locus in type III strains of T. gondii has a large
86 | january 2008 | volume 6 www.nature.com/reviews/micro
© 2008 Nature Publishing Group
disruption in the promoter region that is thought to be
responsible for the massive decrease in its expression
relative to type I and II strains). The hypothesis is that,
in some hosts, high levels of expression of ROP18 are
so problematic to the host or parasite that either the
host or parasite is killed prematurely, with the result
that there is little, if any, transmission. For example,
the highly expressed type II allele of ROP18, if present
in a type III background, generally causes that strain
to be fatal to mice46. Hence, the promoter disruption
of ROP18 might have been necessary for T. gondii to
productively infect mice, whereas full-on expression
might be needed for the infection of some other inter-
mediate hosts. The differences in the coding region
(as opposed to the promoter) might be evidence for
more subtle changes that are needed for optimization
of the interaction of ROP18 with its respective target
in related host species. Ultimately, the situation might
be somewhat analogous to that of the influenza virus,
in which both immune pressure and host range seem
to play important parts in the evolution of several
of its genes. For example, the haemagglutinin gene of
H3N2 viruses is constantly evolving under selective
pressure from the immune system, but for an H5N1
virus to become transmissible between humans other
mutations may need to occur that allow binding to
the particular receptors that are present in the relevant
human tissue51.
When did these differences arise? To answer this
question, additional sequences of ROP16 and ROP18
genes, from many more strains that infect diverse
hosts and, therefore, have diverse ecological niches,
are clearly needed. It is possible that the extremely
large number of coding-sequence differences are a
vestige of a long evolutionary time period, during
which time the sequences became more divergent
as the genes were optimized to different niches. The
disruption of the promoter might be a more recent
event that in one stroke expanded the parasite’s host
range to accommodate the emergence of a new niche,
which, perhaps, is related to human migrations. One
scenario might be that an ancestral North American
strain that experienced the promoter disruption sud-
denly became a productive infector of Norwegian roof
rats or European house sparrows, both of which were
introduced to North America only in the past few cen-
turies. Genotyping of some of the currently most com-
mon strains has shown that one or two matings can
have a dramatic impact on the population biology of
T. gondii 52 and that recent mixing of gene pools from
distant geographic locations has probably occurred;
South America appears to be the source of some of
the greatest diversity53. Such matings may have given
rise to recombinant strains that have just the right mix
of crucial rhoptry proteins, which has enabled the
productive infection of hosts that were previously not
susceptible to this parasite.
Clinical implications of rhoptry biology
A detailed understanding of rhoptry-protein functions
could have profound clinical implications that are well
 REVIEWS

beyond the usual opportunities for the development Conclusions

of new vaccines or drugs to block parasite metabo-
lism. A compound that interferes with assembly or
migration of the MJ, for example, would clearly be
lethal to these obligate intracellular parasites. Much
of the MJ machinery is conserved in the malaria
parasite9,30, so the benefit of such a drug could also
extend to treating this worldwide scourge. ROP16
and ROP18 are kinases that are injected into the host
cell, where their different allelic forms substantially
alter the host–pathogen interaction18,41,46,47. This find-
ing might be key to appropriate decision making if
treating severe ocular toxoplasmosis with steroids that
suppress inflammation54. On the one hand, if infec-
tion is with a strain in which the ROP16 allele causes
an excessive IL-12 response, dampening of inflamma-
tion might be clinically appropriate to prevent damage
to the eye itself. On the other hand, suppressing the
immune response to a strain that expresses a ROP16
allele, which is associated with a weak IL-12 response
(and therefore is naturally producing little inflamma-
tion), might result in an inadequate immune defence,
leading to uncontrolled parasite growth. Knowing
which strain is infecting a patient, therefore, might
allow future clinicians who are armed with a refrigera-
tor full of immune-modulators (for example, recom-
binant cytokines), to tweak the immune response in
the right direction and to the right degree.
Rhoptries contain many of the key molecules that are
used for parasite entry into host cells and subversion of
host functions. The MJ, which contains four known pro-
teins, AMA1, RON2, RON4 and RON5, and probably
several other proteins that have yet to be identified, plays
a central part during invasion. Determining how these
various molecules interact to create the MJ is, therefore,
of great importance to understanding how the parasi-
tophorous vacuole forms. Such knowledge is also likely
to yield some novel findings about interactions between
membranes, as migration of a circular ring of contact
between two membranes without fusion is an unusual
process in biology. The ROP2 family (which includes
ROP16 and ROP18) is extensive and we have almost no
understanding of how this large family of proteins inter-
acts with itself, let alone other host or parasite proteins.
Clearly, based on their high sequence divergence and
role in virulence they are crucial to the host–pathogen
interaction, but the molecular details remain to be dis-
covered. Even those members of the ROP2 family that
seem to have lost kinase activity might be important and
could mediate an effect by regulating the activity of those
members that retain the ability to phosphorylate other
proteins. The prediction that rhoptries contain many
of the molecules that are key to an intracellular lifestyle
is proving correct and recent results are a tantalizing
glimpse of an exciting future.

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Acknowledgements
We thank J. Boyle, P. Bradley, M. Lebrun, M. Reese and
J. Saeij for critical reading of the manuscript and access to
unpublished information. J.C.B. is supported by grants from
the National Institutes of Health (RO1 AI21423 and
AI75473) and the Ellison Medical Foundation and J.F.D. is
supported by grants from the Centre National de la Recherche
Scientifique (UMR5539 and ANR 06-MIME‑024‑01).
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=geneomeprj
Escherichia coli | Plasmodium falciparum | Toxoplasma gondii
GenBank: http://www.ncbi.nlm.nih.gov/Genbank/index.html
RON1 | RON2 | RON3 | RON4 | ROP1 | ROP2A | ROP4 | ROP5 |
ROP6 | ROP7 | ROP8 | ROP9 | ROP10 | ROP11 | ROP12 |
ROP13 | ROP14 | ROP15 | ROP16 | ROP17 | ROP18 | TgNHE2 |
TgPP2C-hn |TgSUB2 | Toxofilin | Toxopain1
FURTHER INFORMATION
John C. Boothroyd’s homepage: http://med.stanford.edu/
profiles/John_Boothroyd/
ToxoDB4.2: http://www.toxodb.org/toxo/home.jsp
SUPPLEMENTARY INFORMATION
See online article: S1 (movie)
All links are active in the online pdf
Correspondence
From ‘perfect mix’ to ‘potion
magique’ — regulatory T cells and
anti-inflammatory cytokines as
adjuvant targets
Jagadeesh Bayry*‡§, Darren R. Flower||, David F Tough¶ and Srini V. Kaveri*‡§
The efficacy of vaccines can be greatly
improved by the addition of adjuvants, which
enhance and modify immune responses.
Historically, adjuvants have been discovered
empirically by using experimental models.
Unfortunately, many adjuvants are associated
with side effects that make them unsuitable
for use in humans. At present, few adjuvants
are available, and of these the adjuvant that is
most commonly used, alum, induces immune
responses that are suboptimal in several
respects. There is an obvious need for the
development of improved adjuvants that can
induce cell-mediated responses in addition to
antibodies.
Recent advances in basic immunology have
revealed the importance of innate pathogen-
recognition receptors in shaping the adaptive
immune response. The Review by Bruno Guy, in
the July issue of Nature Reviews Microbiology 1,
eloquently discusses how Toll-like receptor
(TLR) and non-TLR innate-receptor signal-
ling by antigen-presenting cells (APCs) can
be used to enhance the immunogenicity of
vaccines. Although the activation of APCs
by innate receptors represents a promising
approach to adjuvant development, we wish to
draw attention to an alternative strategy, which
could be used in conjunction with TLR ago-
nists to optimize adjuvant activity. Specifically,
we propose that selective interference with the
activity of regulatory T (TReg) cells and suppres-
sive cytokines could be used to boost responses
to poorly immunogenic vaccines.
Naturally occurring CD4+CD25+ TReg cells
are crucial for the induction and maintenance
of self-tolerance and are present in peripheral
tissues, such as the skin and gut, under nor-
mal, non-inflamed conditions2. TReg cells not
only suppress self-antigen-specific immune
responses, but also negatively regulate immune
responses against foreign antigens, including
those driven by TLR-stimulated APCs3–6. TReg
cells can inhibit initial T‑cell activation and
downregulate ongoing immune responses,
which suggests that they act both at the site
of initiation of immune responses (secondary
nature reviews | microbiology www.nature.com/reviews/micro
lymphoid organs) and at sites of inflamma-
tion; part of the regulatory activity of TReg
cells is related to their ability to modify APCs
such as dendritic cells (DCs)3–6. Notably, TReg
cells themselves have been reported to express
several TLRs, including TLR4, TLR5, TLR7
and TLR8, and stimulation by TLR ligands
can enhance the suppressive functions of TReg
cells7–9. These results suggest that although
TLR stimuli can activate APCs, such signals
might also enhance the suppressive func-
tions of TReg cells. Although this mechanism
is probably important in controlling inflam-
matory responses in the context of infection,
such concomitant suppression might limit
the generation of effective immune responses
after administration of poorly immunogenic
vaccines.
The idea that limiting TReg cell activity at
the time of vaccination is an effective way
of enhancing immune responses has been
confirmed experimentally. Evidence for this
has typically come from studies in which TReg
cells are depleted using anti-CD25 mono-
clonal antibodies. Animals that are depleted
of TReg cells show markedly superior primary
and memory CD8+ T‑cell responses to both
virus infection and vaccines10,11. Although
such experiments provide a proof of princi-
ple, it is unlikely that this approach of TReg cell
depletion would be of clinical use, as it has
been associated with adverse consequences
such as localized autoimmune disease12.
Conversely, the transient inhibition of TReg
cell function might be ideal for enhancing the
immune response to vaccines. One strategy
would be to block the trafficking of TReg cells
to the site of vaccination. TReg cells express
the chemokine receptors CCR4 and CCR8
and migrate in response to the chemokines
CCL17 and CCL22. There is evidence to sug-
gest that the secretion of these chemokines by
DCs can attract TReg cells to the site of inflam-
mation. Further, blocking CCL22 by mono-
clonal antibodies in vivo has been shown to
significantly reduce TReg cell trafficking13.
Similarly, the blockade of vascular endothelial
© 2008 Nature Publishing Group
L i n k t o O r i g i n a l a rt i c l e
L i n k t o A u t h o r ’ s r e p ly
growth factor has also been shown to reduce
the migration of TReg cells14. Consequently, the
inhibition of TReg cell migration at the time
of vaccination — by blocking chemokines or
growth factors through the use of antibod-
ies or small-molecule antagonists — would
alleviate the suppressive effects of TReg cells
during the initiation of the immune response
and allow for a greater response to the
vaccine15.
Cytokines are key regulators of the
immune system that shape innate and adap-
tive immune responses. Because cytokines
might have an adjuvant-like effect, research-
ers have attempted to use them to manipu-
late the immune response to vaccination16.
Notably, the maturation-associated signal-
ling of DCs (including that induced by TLR
ligands) also leads to the production of
anti-inflammatory cytokines such as inter-
leukin-10 (IL-10). Importantly, suppression
by IL-10 can have a significant effect on the
outcome of the adaptive immune response
to vaccination. Fms-like tyrosine-kinase‑3-
based immunoprophylaxis against infection
has been shown to be improved by adjuvant
treatment with anti-IL-10 antibody 17. In
this study, a single injection of anti-IL-10
antibody was associated with an increase
in early IL-12 and interferon-γ production
from innate and adaptive immune cells. This
experimental model suggests that neutral-
izing monoclonal antibodies to IL-10 also
have a potential application in vaccinology.
Together, these findings suggest that the
use of combination adjuvants, which aim to
both activate innate immune mechanisms
and interfere with suppressive mechanisms,
will achieve effective APC activation and
generate robust responses to vaccines.
*Centre de Recherche des Cordeliers, Equipe 16,
Immunopathology and Therapeutic Immunointervention,
Université Pierre et Marie Curie–Paris 6, UMRS872,
15 rue de l’Ecole de Médicine, Paris F‑75006, France.
Université Paris Descartes, UMRS872 Paris
‡
F‑75006, France.
§
INSERM, U872, Paris F‑75006, France.
The Jenner Institute, University of Oxford, Compton,
||
Berkshire RG20 7NN, UK.
¶
Target Discovery, RI CEDD, GlaxoSmithKline, Gunnels
Wood Road, Stevenage SG1 2NY, UK.
Correspondence to J.B.
e-mail: jagadeesh.bayry@crc.jussieu.fr
doi:10.1038/nrmicro1681-c1
1. Guy, B. The perfect mix: recent progress in adjuvant
research. Nature Rev. Microbiol. 5, 505–517 (2007).
2. Miyara, M. & Sakaguchi, S. Natural regulatory T cells:
mechanisms of suppression. Trends Mol. Med. 13,
108–116 (2007).
3. Oldenhove, G. et al. CD4+CD25+ regulatory T cells
control T helper cell type 1 responses to foreign
antigens induced by mature dendritic cells in vivo.
J. Exp. Med. 198, 259–266 (2003).
Correspondence
4. Tang, Q. et al. Visualizing regulatory T cell control of
autoimmune responses in nonobese diabetic mice.
Nature Immunol. 7, 83–92 (2006).
5. Houot, R., Perrot, I., Garcia, E., Durand, I. &
Lebecque, S. Human CD4+CD25high regulatory T cells
modulate myeloid but not plasmacytoid dendritic cells
activation. J. Immunol. 176, 5293–5298 (2006).
6. Bayry, J., Triebel, F., Kaveri, S. V. & Tough, D. F. Human
dendritic cells acquire a semimature phenotype and
lymph node homing potential through interaction with
CD4+CD25+ regulatory T cells. J. Immunol. 178,
4184–4193 (2007).
7. Caramalho, I. et al. Regulatory T cells selectively
express toll-like receptors and are activated by
lipopolysaccharide. J. Exp. Med. 197, 403–411
(2003).
8. Crellin, N. K. et al. Human CD4+ T cells express TLR5
and its ligand flagellin enhances the suppressive
capacity and expression of FOXP3 in CD4+CD25+
T regulatory cells. J. Immunol. 175, 8051–8059
(2005).
nature reviews | microbiology www.nature.com/reviews/micro
9. Lewkowicz, P., Lewkowicz, N., Sasiak, A. &
Tchorzewski, H. Lipopolysaccharide-activated
CD4+CD25+ T regulatory cells inhibit neutrophil
function and promote their apoptosis and death.
J. Immunol. 177, 7155–7163 (2006).
10. Suvas, S., Kumaraguru, U., Pack, C. D., Lee, S. &
Rouse, B. T. CD4+CD25+ T cells regulate virus-specific
primary and memory CD8+ T cell responses. J. Exp.
Med. 198, 889–901 (2003).
11. Toka, F. N., Suvas, S. & Rouse, B. T. CD4+ CD25+
T cells regulate vaccine-generated primary and
memory CD8+ T‑cell responses against herpes
simplex virus type 1. J. Virol. 78, 13082–13089
(2004).
12. Sutmuller, R. P. et al. Synergism of cytotoxic T
lymphocyte-associated antigen 4 blockade and
depletion of CD25+ regulatory T cells in
antitumor therapy reveals alternative pathways
for suppression of autoreactive cytotoxic T
lymphocyte responses. J. Exp. Med. 194, 823–832
(2001).
© 2008 Nature Publishing Group
13. Curiel, T. J. et al. Specific recruitment of regulatory
T cells in ovarian carcinoma fosters immune privilege
and predicts reduced survival. Nature Med. 10,
942–949 (2004).
14. Li, B. et al. Vascular endothelial growth factor
blockade reduces intratumoral regulatory T cells and
enhances the efficacy of a GM‑CSF‑secreting cancer
immunotherapy. Clin. Cancer Res. 12, 6808–6816
(2006).
15. Johnson, Z., Schwarz, M., Power, C. A., Wells, T. N. &
Proudfoot, A. E. Multi-faceted strategies to combat
disease by interference with the chemokine system.
Trends Immunol. 26, 268–274 (2005).
16. Salerno-Goncalves, R. & Sztein, M. B. Cell-mediated
immunity and the challenges for vaccine development.
Trends Microbiol. 14, 536–542 (2006).
17. Das, L., DeVecchio, J. & Heinzel, F. P. Fms-like tyrosine
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Correspondence
From ‘perfect mix’ to ‘potion
magique’ — regulatory T cells and
anti-inflammatory cytokines as
adjuvant targets: reply from Guy
Bruno Guy
The proposal of Bayry and colleagues to
replace adjuvant approaches, such as Toll-like
receptor (TLR) stimulation, with the inhibi-
tion of regulatory T (TReg) cells, or to combine
both strategies, is an attractive one. I would
nevertheless suggest that this strategy would
be best reserved for therapeutic vaccines, in
situations where the negative role of TReg cells
on protective immunity is well established
(for example, in some chronic diseases such
as parasitic diseases or tuberculosis1,2).
As vaccine developers, we can try to mimic
pathogens in their initiation of immune
responses, but we must be cautious of inter-
fering too much with natural regulatory
mechanisms, and in particular the feedback
mechanisms that prevent over-reactions. For
example, TReg cells are essential for preventing
autoimmunity; the role of some TLR agonists
in inducing these disorders has been dis-
cussed3, and any potential risk of this occur-
ing should be limited.
Bayry and colleagues also propose the use
of interleukin-10 (IL-10) suppression, but I
feel this should also be reserved for therapeu-
tic settings. Again, nature itself usually does
things properly, and inflammatory T helper 1
cells have recently been shown to secrete IL‑10
as a feedback mechanism4. Preventing this
nature reviews | microbiology www.nature.com/reviews/micro
L i n k t o O r i g i n a l a rt i c l e
L i n k to I n i t i a l c o r r e s p o n d e n c e
feedback could lead to over-reactive inflamma-
tory responses. Although the goal, target and
mechanism were different, the TGN1412 ‘dis-
aster’ showed us that interfering directly with
regulatory mechanisms can be dangerous.
In conclusion, the approaches proposed
by Bayry and colleagues could be power-
ful but the outcomes are difficult to predict
precisely. For this reason, I would be cautious
about using them in the routine vaccination of
healthy infants (where even rare side-effects
are not acceptable) and would reserve them,
at least initially, for use in therapeutic vaccina-
tion against cancer5, or chronic infections, for
which the benefits might outweigh the risks.
Research Department, sanofi pasteur, Campus
Merieux, Marcy l’Etoile 69280, France.
e-mail: bruno.guy@sanofipasteur.com
doi:10.1038/nrmicro1681-c2
1. Belkaid, Y., Sun, C. M. & Bouladoux, N. et al.
Parasites and immunoregulatory T cells. Curr. Opin.
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2. Scott-Browne, J. P. et al. Expansion and function of
Foxp3-expressing T regulatory cells during
tuberculosis. J. Exp. Med. 204, 2159–2169 (2007).
3. Marshak-Rothstein, A. Toll-like receptors in systemic
autoimmune disease. Nature Rev. Immunol. 6,
823–835 (2006).
4. Trinchieri, G. Interleukin‑10 production by effector
T cells: Th1 cells show self control. J. Exp. Med. 204,
239–243 (2007).
5. Curiel, T. J. Tregs and rethinking cancer immunotherapy.
J. Clin. Invest. 117, 1167–1174 (2007).
© 2008 Nature Publishing Group