Update TRENDS in Parasitology Vol.19 No.
2 February 2003
| Research Focus
Babesiosis: persistence in the face of adversity
David R. Allred
Dept of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA
Many babesial parasites establish infections of long
duration in immune hosts. Among different species, at
least four mechanisms are known that could facilitate
evasion of the host immune response, although no one
species is (yet) known to use them all. This update
strives to illustrate the ramifications of these mechan-
isms and the interplay between them.
Babesia spp. are a diverse group of tick-borne, obligate,
intraerythrocytic Apicomplexan parasites infecting a wide
variety of organisms. Infection of a vertebrate host is
initiated by inoculation of sporozoite stage parasites into
the bloodstream during the taking of a bloodmeal. Most
babesial sporozoites directly invade circulating erythro-
cytes without a tissue stage of development [1]. A few,
notably Babesia equi [2] and Babesia microti [3], first
invade lymphocytes where they form motile merozoites,
which then invade erythrocytes. Although this undoubtedly
affects their interactions with the host, any effects on
immune evasion are at present unknown. Once erythro-
cyte invasion occurs, a seemingly perpetual cycle of
asexual reproduction is established, despite the rapid
development of a strong immune response [4].
During the acute babesial infection, the host may
become severely ill. Typically, the infected host can suffer
high fevers, severe anemia, hemoglobinuria caused by
intravascular hemolysis considerably in excess of that
correlated with parasitemia, lethargy, inappetance, and
sometimes hydrophobia [5]. Coagulatory disturbances are
also a frequent finding [6– 9]. Neurologic sequelae may
occur with some babesial parasites, most notably Babesia
bovis. In the case of B. bovis, this is accompanied by
massive intravascular sequestration of infected red blood
cells (IRBC) carrying mature parasite stages [10– 13].
Despite the potential severity of the acute infection, indi-
viduals who survive generally develop immunity against
disease, but not against infection per se, and could remain
persistently infected [14 –18]. In the case of B. bovis,
infections can persist for years, perhaps even the lifetime
of the animal. Babesial parasites clearly have adapted well
to survival in the hostile environment that is the immune
host. How do they do this?
We understand very little about the mechanisms used
by these parasites to survive. However, at least five
different phenomena are known that probably contribute
to parasite survival: (1) rapid antigenic variation (Fig. 1);
(2) cytoadhesion (Fig. 2 and see animation on: http://
archive.bmn.com/supp/part/allred.html) and sequestra-
tion; (3) binding of host proteins to the IRBC surface; (4)
Corresponding author: David R. Allred (allredd@mail.vetmed.ufl.edu).
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51
the monoallelic expression of different members of multi-
gene families; and (5) establishment of a poorly understood
transient immunosuppression [19 –21]. The relative con-
tributions of the individual phenomena are not known, nor
have definitive demonstrations yet been made that any
of these directly contributes to survival. Here, only brief
discussions of specific examples will be presented, along
with an attempt to put these phenomena into perspective
relative to the establishment of persistent infection.
Antigenic variation
The first evidence for antigenic variation in babesial para-
sites was obtained with Babesia rodhaini [22] and later
with B. bovis [23]. The results of these studies strongly
suggested that fundamental changes had occurred in the
antigenicity of protective antigen(s) in the surviving para-
site populations, and demonstrated a probable association
between recognition of the variant antigen and immune
protection. Unfortunately, the parasite populations were
(a) ves1αA ves1αB ves1αC
...
... ...
...
(b)
...
...
ves1α site of transcription
(i) Recombination with ves1αC
...
...
(ii) Recombination with ves1αA
...
...
(iii) Recombination with ves1αB
...
...
TRENDS in Parasitology
Fig. 1. Antigenic variation in Babesia bovis appears to proceed via a segmental
gene conversion mechanism. In this mechanism, sequences are duplicated from
donor gene copies [a chromosomal segment containing donor genes (orange, pur-
ple and green boxes) is shown in (a)] into an actively transcribed variant erythro-
cyte surface antigen (ves) 1a gene (ves1a site of transcription, yellow box) in (b).
With repeated instances of segmental gene conversion [presumably only one per
generation, e.g. (bi), (bii), (biii)], the actively transcribed ves1a gene becomes pro-
gressively altered from the original, whereas the donor gene copies appear to
remain unaltered. The actual mechanism by which duplicated sequences are trans-
ferred is not known. This site of transcription is shown in a hypothetical placement
near the telomeric end (shown as boxed red and black repeat units). Hypothetical
promoters are indicated by black flags and hypothetical telomere terminal struc-
tures are represented by pink boxes after the repeat units.
52 Update TRENDS in Parasitology Vol.19 No.2 February 2003

Key:

RBC

Ring-stage IRBC
(a) (c)

Trophozoite-stage IRBC that has already made

(b) surface antigens
(d) To Meront-stage IRBC that is expressing a full complement
spleen of surface antigens
Meront-stage that is not expressing surface antigen
or a non-adhesive isoform
TRENDS in Parasitology

Fig. 2. Cytoadhesion of Babesia bovis. Babesia bovis-infected red blood cells (IRBC) can adhere to the capillary endothelium of blood vessels of various tissues, a behavior
that could be abrogated by host antibodies recognizing parasite antigens on the IRBC surface. (a) Babesia bovis IRBC carrying ring stages or mature stages (trophozoites or
meronts) that are expressing non-adhesive surface molecules remain in the peripheral circulation, and will pass through the spleen. Whereas ring-stage IRBCs will prob-
ably survive splenic passage, IRBC carrying mature parasites will be removed and destroyed as a result of antibody recognition of the antigens expressed on IRBC surface.
(b) Babesia bovis IRBC carrying mature parasites that are expressing adhesion-competent forms of proteins exported to the IRBC surface (probably VESA1; shown as green
triangles) will adhere to the endothelium of the capillaries and post-capillary venous circulation (cytoadhesion). These parasites will mature in the deep microvasculature
and release their merozoite stages there. (c) Babesia bovis IRBC expressing different isoforms of the adhesive molecules on the IRBC surface (shown as green U-shaped
molecules) will bind to different receptors and endothelial cells. This could account for apparent tissue tropism in this parasite, including that occurring within the brain. (d)
The development of antibodies (shown as Y-shaped molecules) by the immune host is thought to be capable of preventing cytoadhesion and of causing already-bound
IRBCs to be released from the endothelium. When this occurs, the IRBC are probably susceptible to splenic removal. Note these antibodies recognize the ligands from (b),
but have no effect on IRBC expressing the ligands from (c). For animated version, go to: http://archive.bmn.com/supp/part/allred.html.

not clonal, precluding rigorous interpretation of these
experiments, and the identities of the variant antigen(s) in
B. rodhaini remain unknown.
In contrast to the situation with B. rodhaini, a variant
antigen expressed by B. bovis has been identified. A size-
polymorphic, parasite-derived antigen was identified on
the B. bovis IRBC surface. This antigen was recognized in
an isolate-specific manner during live-cell immunofluor-
escence and surface-specific immunoprecipitation assays
[24]. The application of these assays to parasites recovered
at different times from a calf, infected once with a clonal
B. bovis line, revealed that the isolate-specific antigen
underwent rapid size and antigenic variation [16]. The
putative variant antigen was subsequently confirmed
through the use of monoclonal antibodies (mAb), and was
named the variant erythrocyte surface antigen (VESA) 1
[25]. This antigen migrates on sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) as a
doublet, ranging in mass between 105 kDa and 135 kDa,
among different variants and isolates. Recently, the ves1a
multigene family encoding the larger subunit (VESA1a)
was identified [26], and preliminary estimates suggest a
minimum of $ 50 gene copies. The molecular mechanisms
involved in expression of variation are only beginning to be
worked out, but all evidence to date is consistent with
progressive, segmental gene conversion playing a key role
[26] (Fig. 1). Some ves1a genes can donate sequences to
the transcribed gene copy, while apparently remaining
unaltered themselves ([26]; B. Al-Khedery et al., unpub-
lished). It is not yet clear whether all ves1a genes can
participate in the phenomenon of gene conversion.
Coupled with a similarly polymorphic VESA1b subunit
[16,24,25], the potential capacity of this parasite for
presentation of unique VESA1 antigens is enormous.
Cytoadhesion and sequestration
The spleen is a remarkable organ in its ability to recognize
and remove damaged cells from circulation. It could be
http://trepar.trends.com
aided by the presence of bound antibodies and, perhaps
complement, on the damaged cell’s surface. Accordingly,
passage through the spleen is a journey to be avoided by
parasitized erythrocytes. The ability to avoid splenic pas-
sage has been observed in Babesia canis [27], the Babesia
Washington state isolate 1 (WA1) [28] and B. bovis [29,30],
although it is likely that different mechanisms are used to
achieve sequestration. It has been suggested that B. canis
becomes trapped in the deep vasculature through general-
ized vasodilation and hypotensive pooling of blood. Under
these conditions, IRBC might fail to circulate, becoming
trapped in local coagulatory masses where they undergo
proliferation [27]. Nothing is currently known of the
mechanism(s) used by the WA1 isolate to sequester, and the
act of sequestration itself could be host-specific [28,31].
By contrast to B. canis, B. bovis sequesters in the
microvasculature through specific binding of IRBC to the
capillary and post-capillary endothelium via knob-like
protrusions of the IRBC membrane (Fig. 2 and http://
archive.bmn.com/supp/part/allred.html) [10,11,32]. This
binding (cytoadhesion) and sequestration is associated
with the severe and frequently lethal cerebral form of
bovine babesiosis [10 –13,30]. Using an in vitro assay of
cytoadhesion to characterize this phenomenon, it was
shown that parasite-synthesized components must be
present for binding to occur [33]. Further, strong circum-
stantial evidence was found for mediation of this phenom-
enon by the VESA1 antigen [34]. Clear precedents exist, in
the Plasmodium falciparum erythrocyte membrane pro-
tein (PfEMP) 1 [35,36], and the M proteins of Streptococcus
pyogenes [37], for mediation of pathogen adhesion to host
cells by highly variant components. The endothelial
receptor for B. bovis cytoadhesion is not known, but cur-
rent data do not support a significant role for compatibility
determinant (CD)36 [33], the major P. falciparum receptor.
It has recently been hypothesized that sequestration
results in an enhanced parasite susceptibility to killing by
reactive nitrogen oxides due to unloading at reduced oxygen
 Update TRENDS in Parasitology
tension of erythrocytes laden with nitric oxide (NO) [38]. It
was previously shown that B. bovis-stimulated macrophages
produce NO, and that in vitro parasite killing by NO can be
achieved [39]. However, a diminished capacity for macro-
phage NO production has been observed under reduced
oxygen tension [40], and other studies demonstrate NO
consumption by oxyhemoglobin, with the release only of
nitrate and methemoglobin [41]. Thus, sequestration might
serve to enhance parasite survival by preventing localized
circulation of NO-carrying erythrocytes and maintaining
local hypoxia. Clearly, all the ramifications of cytoadhesion
are not yet understood, but the ability to sequester and not
suffer splenic removal is probably a very important
mechanism of long-term survival by this parasite.
Binding of host proteins to the IRBC surface
Not all babesial parasites appear to undergo antigenic
variation, and most do not cytoadhere or sequester. The
bovine parasite, Babesia bigemina, is a good example.
Although B. bigemina expresses parasite-derived antigens
on the IRBC surface, the antigens appear to be isolate-
common and stable over the course of infection [42].
However, B. bigemina IRBC bind immunoglobulin (Ig) M
on the IRBC surface, in a non-immunospecific reaction [43].
Presentation of IgM molecules, bound through the Fc region
with the antigen recognition domain facing the plasma,
clearly represents a mechanism with the potential to hide
this parasite from immune recognition. It is possible that the
invariant IRBC surface antigens mediate IgM binding, but
such a connection has not been made. The number of IgM
bound on individual cells is also not known, but if sufficiently
dense could render these antigens inaccessible to specific
antibodies and leave IRBC ‘invisible’ to circulating immune
effector cells. How this would affect the interaction of IRBC
with the spleen is less clear, but might reduce susceptibility
to splenic clearance as well, and could help to explain the
persistence (up to two years [14]) of this parasite.
Monoallelic expression of members of multigene
families
Babesia microti is by far the most common cause of human
babesiosis [44]. Similar to many babesial parasites, this
species is capable of persisting in the immune host, even
after non-sterilizing chemotherapy [18]. The mechanisms
used to survive in individual hosts have not yet been
elucidated, but a recent clue to population survival might
have emerged. Using immune sera from individuals who
had recovered from babesiosis to screen complementary
DNA (cDNA) libraries, three immunodominant antigen
gene families were discovered [45]. In a limited study
monitoring expression of the Babesia microti MN1 strain
(bmn1) gene family, it appeared that a single member was
transcribed. Further, it was revealed that different alleles
were expressed by parasites infecting patients from
different geographical areas. By contrast, the same allele
was expressed in several patients from a small area of
transmission, and this was stable upon transmission to
hamsters [46]. These results make it unlikely that the
bmn1 gene family is involved in antigenic variation as was
suggested by the authors. However, the considerable
diversity within this gene family and its (apparently)
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Vol.19 No.2 February 2003 53
subtelomeric location would be consistent with frequent
recombination events. If bmn1 gene products are associ-
ated with immunoprotection, this allelic diversity might
provide the parasite with an opportunity to establish
mixed infections in immune hosts already primed by
exposure to other members of the family. In such a
scenario, the temporal nature might lie between the very
rapid change of antigenic variation and the relatively slow
evolution associated with allelic polymorphism. Despite
the attractiveness of this possibility, the association of
allelic polymorphism per se with immune evasion is not
always clear. For example, in B. bigemina, four alleles of
the merozoite surface antigen (msa) 2 multigene family
have been described, the products of which are immuno-
logically non-crossreactive [47]. At least three of the four
alleles are co-expressed on individual merozoite- and
sporozoite-stage parasites, yet there is no additive effect
on inhibition of invasion (or binding) by simultaneous
exposure to antibodies against all four gene products [48].
Interplay of these phenomena
Currently, nothing is known of interplay between antigen
masking or monoallelic expression of invariant multigene
families and antigenic variation or cytoadhesion, if such
interaction occurs. Strong circumstantial evidence sug-
gests that the rapidly variant VESA1 antigen on the
B. bovis IRBC surface serves as a parasite-derived ligand
mediating cytoadhesion [34]. One obvious potential out-
come of this is that an in vivo switch in an antigenic
phenotype, selected by immune recognition of the pre-
viously expressed VESA1a isoform, probably results in
alterations of the adhesive phenotype (Fig. 2 and http://
archive.bmn.com/supp/part/allred.html). This effect was
observed in vitro, using an assay of B. bovis cytoadhesion
to bovine brain endothelial cells [34]. Further, the C9.1
line-derived mAb, 3F7.1H11, which also reacts with the
cytoadhesive CD7 line (as a result of selection for this
trait), is capable of both blockage and reversal of in vitro
cytoadhesion, with great efficacy [34]. In addition, the
adhesion specificity of different clonal B. bovis lines for
different clonal bovine brain endothelial cell lines varies
(R.M. O’Connor and D.R. Allred, unpublished), suggesting
either the recognition of different endothelial receptors,
varied affinities for shared receptors, or both. Thus, in vivo
recognition and elimination of one parasite population,
with expansion of another, probably results in the selection
of parasites with differing adhesive phenotypes and
perhaps tissue tropism (Fig. 2 and http://archive.bmn.
com/supp/part/allred.html). Given the tissue-specific pres-
entation of endothelial receptor motifs [49], a protective
immune response to one population could inadvertently
result in relocation of the infection focus, modifying the
potential pathology, and perhaps delaying the initiation of
a response to surviving parasites. Early studies on the
histopathology of B. bovis infection reported widely
varying parasitemias for parasites localized in different
tissues, consistent with this possibility [10,11,32].
Conclusions
Babesia spp. are a seriously understudied group of parasites.
However, at least five mechanisms have been identified
54 Update TRENDS in Parasitology
which might contribute to their evasion of host immunity.
Antigenic variation, cytoadhesion/sequestration, host-pro-
tein binding, and induction of immunosuppression probably
facilitate persistence in the individual immune host.
Monoallelic expression of different members of a multi-
gene family might facilitate multiple infections of immune
hosts, and population dispersal in endemic areas.
Acknowledgements
D.R.A. gratefully acknowledges Basima Al-Khedery, Julie Crabtree,
Roberta O’Connor, Kristi Warren and Rachel Jervis for their assistance.
This work was supported by grants from the United States Dept. of
Agriculture (#2001 – 35204 – 10144) and the American Heart Association
(#0051422B).
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Characterization and role of protozoan parasite
proteasomes
André Paugam1, Anne-Laure Bulteau2, Jean Dupouy-Camet1, Claudine Creuzet1 and
Bertrand Friguet2
1
Laboratoire Signalisation et Parasites (EA 3623), Université Paris 5, C.H.U. Cochin, 27, rue du Faubourg Saint Jacques 75014 Paris,
France.
2
Laboratoire de Biochimie et de Biologie du Vieillissement (EA 3106) Université Paris 7-Denis Diderot, 2, place Jussieu, 75251, Paris,
France.
The proteasome, a large non-lysosomal multi-subunit
protease complex, is ubiquitous in eukaryotic cells. In
protozoan parasites, the proteasome is involved in
cell differentiation and replication, and could therefore
be a promising therapeutic target. This article reviews
the present knowledge of proteasomes in protozoan
parasites of medical importance such as Giardia,
Entamoeba, Leishmania, Trypanosoma, Plasmodium
and Toxoplasma spp.
The proteasome (for reviews, see Refs [1,2]) has important
roles in protein turnover in both the cytosol and the
nucleus of eukaryotic cells. It is the centrepiece of the
non-lysosomal, ubiquitin-dependent protein degradation
pathway. In this pathway, most substrates are first
marked for degradation by covalent linkage to multiple
ubiquitin molecules. Ubiquitin-conjugated proteins are
then rapidly degraded by the 26S proteasome, a 2000-kDa
ATP-dependent proteolytic complex [3]. The proteasome is
a dynamic structure built in a modular manner. The 26S
proteasome comprises a barrel-shaped 20S catalytic core
complex capped at one or both ends by a 19S complex, also
called the proteasome activator (PA)700, which confers
substrate specificity and regulation (Fig. 1). Another regu-
lator complex termed 11S (or PA28) can replace 19S and
activate the proteolysis of short peptides. Proteasomes
have several distinct peptidase activities, chief among
which are chymotrypsin-like and trypsin-like activities.
The main roles of the proteasome include: (1) proteolysis of
abnormal, misfolded or improperly assembled proteins;
and (2) control of cell cycle by selective degradation of
regulatory proteins such as transcription factors and
cyclins. Proteasomes are also involved in a wide range
of biological functions such as cell differentiation, stress
responses, metabolic adaptation and cellular immune
Corresponding author: André Paugam (andre.paugam@cch.ap-hop-paris.fr).
http://trepar.trends.com
Vol.19 No.2 February 2003 55
specific antibodies inhibit attachment and invasion of erythrocytes.
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1471-4922/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S1471-4922(02)00065-X
responses. The structure of the proteasome has been
recently described. The 700-kDa proteasome complex
comprises 28 subunits, which have similar molecular
masses (ranging from 20 to 35 kDa), but a wide range of
isoelectric points (4.5 to 8.7). Under the electron micro-
scope, these subunits appear to be arranged as a hollow
cylinder composed of four stacked rings, each containing
seven subunits (Fig. 2) and a central channel with three
large cavities. The two outer cavities are located at the
interfaces between the a and b rings. The third cavity
is located at the center of the complex and is formed by the
b rings (Fig. 1).
The primitive archebacterial proteasome purified from
Thermoplasma acidophilum possesses the same quatern-
ary structure as the eukaryotic proteasome, but has only
two non-identical subunits (a and b). a-subunits form
the two outer rings and b-subunits the two inner rings
(a7b7b7a7). The proteolytic sites are formed by b-subunits
that carry N-terminal threonine residues faced to the
central cavity of the 20S complex. Contrary to arche-
bacterial proteasome, eukaryotic proteasomes have mul-
tiple types of hydrolytic activities that are due to the
different b-subunits. The outer rings consist of a non-
proteolytic a-subunit that allows translocation into the
central cavity, and conformational interactions between
the 20S complex and the regulatory complex. The type
of regulatory complex varies depending on the function
of the proteasome, for example, 19S and 11S activate
degradation of ubiquitinated proteins and peptides,
respectively. In certain cases, these regulatory complexes
are inducible [11S with interferon (INF)-g] and might only
be found in certain cell types or at specific points of the cell
cycle. Molecular cloning shows intersubunit homology and
remarkable conservation of subunit sequences of a to a
and b to b between Thermoplasma and eukaryotic cells
(i.e. 20S yeast identity varies from 43% to 66%, when
compared with Thermoplasma). All eukaryotic subunits