Article

Chemodetection and Destruction of Host Urea

Allows Helicobacter pylori to Locate the Epithelium
Graphical Abstract Authors
Julie Y. Huang, Emily Goers Sweeney,
Michael Sigal, ..., Calvin J. Kuo, Karen
Guillemin, Manuel R. Amieva

Correspondence
amieva@stanford.edu

In Brief
The signals that direct Helicobacter pylori
to the gastric epithelium have not been
well defined. Huang et al. demonstrate
that H. pylori possesses a sensitive
chemoreception system that couples
simultaneous destruction and detection
of urea gradients emanating from the
human gastric epithelium to find the host
epithelial niche.

Highlights
d H. pylori is attracted to metabolites emanating from the
human gastric epithelium

d Urea is the primary host metabolite that attracts H. pylori

d H. pylori’s high-affinity chemoreceptor TlpB detects

nanomolar amounts of urea

d Urea degradation by bacterial urease facilitates sensing

through TlpB

Huang et al., 2015, Cell Host & Microbe 18, 147–156

August 12, 2015 ª2015 Elsevier Inc.
http://dx.doi.org/10.1016/j.chom.2015.07.002
Cell Host & Microbe

Article

Chemodetection and Destruction of Host Urea Allows

Helicobacter pylori to Locate the Epithelium
Julie Y. Huang,1 Emily Goers Sweeney,2 Michael Sigal,3 Hai C. Zhang,4 S. James Remington,2,5 Michael A. Cantrell,4
Calvin J. Kuo,4 Karen Guillemin,2 and Manuel R. Amieva1,3,*
1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
2Instituteof Molecular Biology, University of Oregon, Eugene, OR 97403, USA
3Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA
4Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
5Department of Physics, University of Oregon, Eugene, OR 97403, USA

*Correspondence: amieva@stanford.edu
http://dx.doi.org/10.1016/j.chom.2015.07.002

SUMMARY gastric epithelium and remains within 25 mm of the stomach sur-

The gastric pathogen Helicobacter pylori interacts
intimately with the gastric mucosa to avoid the
microbicidal acid in the stomach lumen. The cues
H. pylori senses to locate and colonize the gastric
epithelium have not been well defined. We show
that metabolites emanating from human gastric orga-
noids rapidly attract H. pylori. This response is largely
controlled by the bacterial chemoreceptor TlpB, and
the main attractant emanating from epithelia is urea.
Our previous structural analyses show that TlpB
binds urea with high affinity. Here we demonstrate
that this tight binding controls highly sensitive re-
sponses, allowing detection of urea concentrations
as low as 50 nM. Attraction to urea requires that
H. pylori urease simultaneously destroys the signal.
We propose that H. pylori has evolved a sensitive
urea chemodetection and destruction system that
allows the bacterium to dynamically and locally
modify the host environment to locate the epithelium.
INTRODUCTION
Helicobacter pylori colonizes the stomachs of half of the world’s
population, and chronic infection is the strongest risk factor for
developing peptic ulcers and gastric cancer (Marshall and War-
ren, 1984; Parsonnet et al., 1991). The human stomach is one of
the harshest environments that microbes encounter due to its
content of hydrochloric acid, proteolytic enzymes, and rapid
clearance mechanisms (Yang et al., 2013). Despite the fact
that H. pylori is a permanent resident of the stomach, it is not
an acidophile (Bauerfeind et al., 1997) and has evolved strategies
to avoid the acidic environment using motility and chemotaxis
(Salama et al., 2013).
H. pylori actively swims away from the acidic lumen, and to
buffer itself and its local environment during this transit, it pro-
duces abundant amounts of urease, a highly efficient enzyme
that converts urea into ammonia and bicarbonate (Marshall
et al., 1990; Mobley et al., 1995; Scott et al., 1998, 2002). To
establish colonization, H. pylori navigates to the surface of the
face either in the protective mucus layer or directly adhered to
the epithelium (Howitt et al., 2011; Schreiber et al., 2004). The
attached bacteria extract nutrients from the host (Tan et al.,
2011) and utilize the cell surface as a replicative niche where
they form microcolonies (Tan et al., 2009). H. pylori also forms
microcolonies deep within the gastric glands (Howitt et al.,
2011) and interacts with gastric progenitor and stem cells to
induce host pathology (Sigal et al., 2015).
Hydrochloric acid has been shown to be a chemorepellent for
H. pylori (Croxen et al., 2006; Goers Sweeney et al., 2012; Howitt
et al., 2011) and is thought to help H. pylori avoid the gastric
lumen (Schreiber et al., 2004). However, in addition to this impor-
tant signal, it is likely that there are signals emanating from the
gastric epithelium that attract H. pylori to the cell surface. Indeed,
H. pylori was recently shown to swim toward injured epithelia,
suggesting that H. pylori is attracted to host-derived molecules
(Aihara et al., 2014).
In this study, we aimed to determine the mechanism by which
H. pylori locates the epithelium. Using human gastric organoids
and well-characterized epithelial cell lines, as well as video mi-
croscopy to assess rapid chemotactic responses, we show
that H. pylori is attracted within seconds to small amounts of
host metabolites that emanate from polarized epithelia. We
determined the main bacterial chemoreceptor responsible for
this chemoattraction to be TlpB and identified urea as the host
metabolite that attracts H. pylori. Like previous studies, we found
that in a murine model of infection, multiple signals contribute
to colonization since TlpB is not required for establishment
of infection. However, we found that mutants in TlpB are defi-
cient in persistence. In addition, we uncovered a function for
H. pylori’s urease enzyme in facilitating sensitive detection of
urea at concentrations as low as 50 nM. Our findings demon-
strate that microbes that have established intimate associations
with their host, such as H. pylori, are exceptionally fine-tuned to
signals in the host environment to ensure their survival.
RESULTS
H. pylori Is Attracted to Metabolites Emanating from
Polarized Epithelia
To determine whether H. pylori is capable of sensing and
responding to host metabolites emanating from the gastric

Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc. 147
Figure 1. H. pylori Is Attracted to Metabolites Emanating from Human Gastric Organoids and Polarized Epithelial Cells
(A) Confocal reconstruction of a human gastric organoid in 3D culture stained for nuclei and the actin cytoskeleton. Scale bar, 10 mm.
(B) Confocal immunofluorescence section through the surface epithelium of a human stomach sample (left) and an organoid (right) stained for surface mucus
(MUC5AC). Scale bar, 10 mm.
(C) Confocal 3D reconstruction of an organoid infected with H. pylori for 4 hr. Inset shows higher magnification of H. pylori adhered to epithelial junctions. Scale
bar, 10 mm.
(D) Top: conditioned media was collected near the surface of gastric organoids and loaded into the micropipette. Bottom: test substances form a microgradient at
the micropipette tip as illustrated by injection of India ink in water.
(E) Motility tracings of wild-type (WT) H. pylori’s response to a gradient of organoid-conditioned media (top panels) versus non-conditioned media (bottom panels)
at 1 s or 30 s post-injection. Motility tracings are obtained by combining 15 consecutive frames into a single image.
(F) The concentration of bacteria (pixel density) within 60 mm of the micropipette tip is plotted over time. Each point represents the pixel density at a particular time
in each of three independent movies. WT H. pylori’s response to organoid-conditioned media (red) or non-conditioned media (blue) and of DcheW to organoid-
conditioned media (green) are compared. Zero second is defined as the moment the micropipette is introduced into the viewing field. (n = 3 movies per condition).
(G) The responses of WT H. pylori to Caco-2 cell-conditioned media versus non-conditioned media are plotted. Data are displayed as in (F) (n = 3 movies per
condition).

(legend continued on next page)

148 Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc.
epithelium, we used an ex vivo human gastric organoid system
(Bartfeld et al., 2014; Sato et al., 2011). Human gastric organoids
(Figure 1A) recreate important features of the gastric epithelium
such as polarization and differentiation into gastric mucin-pro-
ducing pit cells (Figure 1B). When cultured in Matrigel most orga-
noids develop with an inside-in polarity (Figure 1A). However,
some organoids have an inside-out polarity allowing access to
the apical surface by swimming bacteria (Figure 1B, right panel).
We examined interactions of free-swimming H. pylori with hu-
man gastric organoids and found that the bacteria are able to
swim and adhere to the cell-cell junctions (Figure 1C) as we
have observed in other polarized epithelia infection models
(Tan et al., 2009) and in vivo (Sigal et al., 2015).
To determine if H. pylori can sense molecules diffusing from
organoids, we extensively washed uninfected organoids and
allowed molecules diffusing from the gastric epithelium to condi-
tion DMEM media. We then collected the conditioned media and
tested H. pylori’s swimming response using a technology we
previously developed to study negative chemotaxis to acid
(Howitt et al., 2011). The conditioned media was loaded into a
micropipette (Figure 1D, top panel) connected to a microinjec-
tion system. The micropipette was then lowered into a culture
of rapidly swimming H. pylori (Figure S1A). We created a micro-
scopic gradient with the point source at the micropipette tip and
monitored the bacterial responses to the gradient in real time
using phase-contrast video microscopy (Figure 1D, bottom
panel). By generating movies and quantifying the pixel density
(proxy for bacterial density) within 60 mm from the micropipette
tip (Figure S1B), we found that H. pylori is rapidly attracted to
organoid-conditioned media but not to non-conditioned
DMEM (Figure 1E and Movie S1). This attraction is dependent
on chemotaxis, as a DcheW mutant lacking a core chemotaxis
adaptor protein (Pittman et al., 2001) does not respond to the
gradient (Figure 1F). These data reveal that H. pylori can detect
and respond to minute amounts of metabolites emanating from
the human gastric epithelium.
To determine if the chemoattractants are specific to gastric
cells or also released by other epithelia in culture, we conditioned
media with Caco-2 cells (a human colon adenocarcinoma cell
line) or Madin-Darby Canine Kidney (MDCK) cells grown as
polarized monolayers on Transwell filters. H. pylori is also rapidly
attracted to DMEM conditioned by metabolites that diffuse to the
apical side of polarized epithelia (Figure 1G, Figure S2A, and
Movie S2). A sufficient quantity of chemoattractants is present
to elicit a response as early as 2 hr (Figure S2B). Taken together,
these data suggest that molecules that are generated by or can
diffuse across epithelial cells rapidly attract H. pylori and may
help H. pylori locate the epithelial surface.
H. pylori Chemoattraction to Host Metabolites Is
Dependent on the Chemoreceptor TlpB
Next, we characterized the mechanism by which H. pylori senses
metabolites emanating from the epithelium. We created isogenic
H. pylori mutants lacking each of the four known chemorecep-
(H) The responses of WT H. pylori versus chemoreceptor mutants to Caco-2 cell-conditioned media are plotted. Data are displayed as in (F) (n = 3 movies per
condition). The responses of DtlpA, DtlpC, and DtlpD are not significantly different from each other.
p values for scatter plots indicate significance of time by group interaction via a two-way repeated-measures ANOVA. See also Figures S1 and S2 and Movies
S1 and S2.
Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc. 149
tors—TlpA, TlpB, TlpC, and TlpD (Figure S2C)—and tested
each for its response to cell-conditioned media. All mutants
were rapidly attracted to cell-conditioned media except for the
mutant lacking TlpB (Figure 1H). We confirmed that TlpB is
also required to sense the human gastric organoid-conditioned
media (Figure S2D). TlpB has been reported to detect acidic
pH (Croxen et al., 2006; Goers Sweeney et al., 2012) and the
quorum-sensing molecule, auto-inducer 2 (AI-2) (Rader et al.,
2011). However, both acid and AI-2 are chemorepellents, indi-
cating that we were observing responses to a chemoattractant
sensed by TlpB. TlpB and many other chemoreceptors are in-
serted into the inner membrane with their ligand-binding do-
mains within the periplasm. We previously reported that a
molecule of urea is tightly bound to the Per-ARNT-Sim domain
of the receptor (Goers Sweeney et al., 2012). We had estimated
the apparent KD of TlpB for urea (relative to formamide) to be
approximately 700 nM. The actual affinity for urea is likely to be
much higher, but due to the insolubility of apo-TlpB, it could
not be directly measured. TlpB’s affinity for urea is higher than
many previously reported chemoreceptor-ligand interactions,
which range between 0.1 mM and 1 mM (Foster et al., 1985;
Iwama et al., 1997; Pineda-Molina et al., 2012; Willis and Furlong,
1974). A few examples of high-affinity chemoreceptors with KD
values in the nanomolar range reflect conditions in which the
ligand is present in very low amounts since higher concentra-
tions in the micromolar range would inhibit the chemotactic
response (Pasupuleti et al., 2014). Since urea is present at con-
centrations of 1–5 mM in gastric secretions (Blusiewicz et al.,
2005), an affinity in the nanomolar range would consistently satu-
rate the receptor and abrogate chemotaxis. However, urea is a
common byproduct of cellular metabolism, is found in gastric se-
cretions as a result of paracellular and transcellular diffusion
across the epithelium, and has been previously reported to act
as a chemoattractant to H. pylori in a capillary tube assay (Mizote
et al., 1997; Worku et al., 2004). Therefore, we tested whether
urea was the epithelial metabolite that attracts H. pylori through
TlpB sensing.
H. pylori Directly Senses Urea through Its
Chemoreceptor TlpB
To test whether urea accumulates in the conditioned media over
time, we used a colorimetric urea quantification method (Gindler,
1981). We found that the urea concentration in the cell-condi-
tioned media increases over time, and if we treat the organoid-
or cell-conditioned media with purified Jack Bean urease, we
can reduce the urea concentration below detectable levels (Fig-
ure S3A). We found that urease treatment abrogates the chemo-
tactic response (Figure 2A).
Given that urease treatment abolished the response, we
tested H. pylori’s response to urea directly. We found that a
1 mM urea solution in water is sufficient to attract wild-type
H. pylori in the microgradient assay (water control showed no
response) and confirmed that this urea chemotactic response
is abolished in a DtlpB mutant (Figure 2B and Movie S3).

Figure 2. TlpB Senses Urea Released from the Epithelium
(A) The concentration of bacteria (pixel density) within 60 mm of the micropi-
pette tip was first determined from the scatter plots as those in Figure 1. WT
H. pylori’s response at 4 s pre-injection and 15 s post-injection of organoid-
conditioned media and Caco-2 cell-conditioned media before and after urease
treatment are displayed in the bar graph (n = 3 movies per condition).
(B) The responses of WT H. pylori, DtlpB, DtlpACD, and DcheW to 1 mM
urea microgradient as well as the response of WT H. pylori to 1 mM urea in
urease-treated Caco-2 cell-conditioned media are plotted (n = 3 movies per
condition).
(C) The responses of WT SS1, WT 7.13, WT G27MA, DtlpB G27MA, and
tlpB* G27MA to 1 mM urea microgradient are plotted. Bacterial density (pixel
density) was quantified within a 60 mm radius from the micropipette tip
and shown from 4 s pre-injection to 30 s post-injections. Each point represents
the pixel density at a particular time in each of the movies. (n = 1 movie per
strain).
150 Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc.
Furthermore, TlpB is sufficient in sensing urea since a triple
mutant lacking TlpA, TlpC, and TlpD and expressing only TlpB
still swims toward urea (Figure 2B). Addition of 1 mM urea into
the urease-treated cell-conditioned media restored the chemo-
attraction response (Figure 2B).
We next determined whether other H. pylori strains also
respond to urea. We observed that, indeed, other H. pylori
strains are also attracted to 1 mM urea (Figure 2C). In the more
genetically manipulable strain H. pylori G27MA (Amieva et al.,
2002) we were able to complement TlpB expression by reintro-
ducing the tlpB gene into its native locus. This confirmed that
urea sensing is dependent on TlpB since DtlpB is unable to
respond to urea, but complementation of DtlpB restores the
response (Figures 2C).
Chemotaxis through TlpB Is Important for Persistent
Colonization In Vivo
Given that TlpB is necessary to sense a urea gradient emanating
from the gastric epithelium, we wondered how DtlpB would fare
in vivo in the stomach. Bioinformatic analysis of all published
H. pylori genomes shows that the tlpB gene is present in all
known H. pylori strains, suggesting that the chemoreceptor is
important for life in the human stomach. However, previous
animal experiments have shown a defect in colonization levels
in one model (Croxen et al., 2006) but not in others (McGee
et al., 2005; Rolig et al., 2012; Williams et al., 2007). We therefore
tested an isogenic tlpB deletion mutant in a mouse model of
colonization using the mouse-adapted strain PMSS1 (Arnold
et al., 2011). We conducted a 2-week infection with wild-type
(WT) H. pylori and two independently isolated DtlpB mutants.
At this time point there was no significant difference in the recov-
ered bacterial load in the stomach between WT and DtlpB (Fig-
ure S3B). This is consistent with previously reported data on
colonization levels of single infections at 2 weeks (Rolig et al.,
2012). These results suggest that there may be other signals
in vivo that initially allow H. pylori to reach their preferred niche
and establish colonization.
Because previous studies have reported different colonization
outcomes at later time points (Croxen et al., 2006; McGee et al.,
2005; Williams et al., 2007), we conducted longer infections to
determine if DtlpB was able to persist in the stomach. We found
that at 4 weeks post-infection there was a trend toward a defect
in DtlpB that was not yet significant. At 6 weeks post-infection,
DtlpB showed a significant decrease in the bacterial load in the
stomach compared to WT (Figure S3B). These results suggest
that TlpB is important for H. pylori long-term persistence in the
stomach.
TlpB Is a High-Affinity Chemoreceptor for Urea
Given the unusually high affinity of TlpB for urea, we wished to
determine the mechanism by which TlpB senses urea. The
most abundantly produced protein in H. pylori is a urease
enzyme that efficiently degrades urea into ammonium and
Bars represent the mean. Error bars represent SD. NS indicates no statistical
significance, ***p < 0.001, ****p < 0.0001 (two-way repeated-measures
ANOVA). p value for scatter plot indicates significance of time by group
interaction via a two-way repeated-measures ANOVA. See also Figure S3 and
Movie S3.

bicarbonate. It is therefore possible that TlpB may be indirectly
sensing urea by detecting the byproducts of urea degradation,
or even a pH change produced by the local degradation of
urea. We tested the response of H. pylori to each byproduct
alone at the expected concentrations from the degradation of
1 mM urea as well as a mixture of 10 mM sodium bicarbonate
and 10 mM ammonium chloride. We found that H. pylori did
not respond to these compounds in the microgradient assay
(Figure S3C), suggesting the hypothesis that TlpB directly
senses urea.
We reasoned that if H. pylori senses urea through direct bind-
ing to TlpB, then it should also respond to other urea analogs that
bind within the TlpB urea-binding pocket. The urea analogs hy-
droxyurea, acetamide, and formamide have either one or both
amine groups of urea replaced with another functional group.
Examination of the ligand environment in our previously pub-
lished crystal structures of TlpB containing bound urea or urea
analogs (Goers Sweeney et al., 2012) confirmed that only urea
has a well-defined orientation within TlpB with saturated
hydrogen-bonding capability (Figure 3A). The analogs either
lack certain hydrogen bonds and/or assume multiple orienta-
tions within the binding pocket (Figure S4A).
To gain insight into the relative binding affinities of these ana-
logs to TlpB, we used a thermofluor assay (Nettleship et al.,
2008), which provides estimates for the thermal stability of the
TlpB periplasmic domain in complex with urea or the analogs.
The thermal stability results suggest that the domain binds
Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc. 151
Figure 3. TlpB Detects Urea and Urea Ana-
logs Directly with Sensitivities that Corre-
late with Their Binding Affinities
(A) Crystal structures of urea and urea analog ori-
entations within the TlpB periplasmic domain.
Orange dashed lines indicate hydrogen bonds.
Yellow spheres represent water molecules.
(B) Thermostability of TlpB measured in the pres-
ence of urea or urea analogs using a thermofluor
assay. Center values represent the mean. Error
bars represent SD.
(C) The minimal concentration of urea versus urea
analogs needed to elicit a WT H. pylori chemo-
attraction response defined as a bacterial swarm
with a 60 mm radius from the micropipette tip at
15 s post-injection.
(D) Motility tracings of WT H. pylori’s response
to microgradients of 500 mM urea or urea analogs
at 15 s post-injection. See also Figure S4 and
Movie S4.
urea most tightly, followed by hydroxy-
urea, acetamide, and formamide in order
of decreasing affinity (Figure 3B). Given
this hierarchy of affinities, we then tested
whether H. pylori responds to these ana-
logs and compared the responses to
those obtained using urea. We found
that H. pylori is attracted to all three ana-
logs, with responses that decreased in
agreement with the decreased binding
affinities of the receptor for these com-
pounds (Figures 3C and 3D and Movie
S4). DtlpB cannot sense any of these compounds. Furthermore,
we could inhibit H. pylori’s response to urea by adding exoge-
nous formamide to the bacterial culture (Figure S4B). Since
TlpB’s affinity for formamide is much lower than that for
urea, 25 mM formamide must be present to block H. pylori’s
response to a gradient generated from 1 mM urea. We conclude
that H. pylori senses urea and its analogs through direct
binding to TlpB, and the sensitivity of the chemoattraction
response is directly correlated to the receptor’s affinity for the
chemoattractant.
Next, we asked whether the unusually high affinity of TlpB for
urea could allow the bacterium sensitive detection of this com-
pound. We conducted a dose response experiment and found
that injecting urea via micropipette at concentrations ranging
from 15 mM to 1 mM urea attracted an increasingly larger number
of bacteria (Figure 4A). However, as urea diffuses rapidly from
the micropipette tip and the bacteria respond at least 60 mm
from the point source, the threshold concentration detected by
the bacteria is likely much less than 15 mM. To determine this
threshold concentration, we tested the swimming response of
H. pylori to a pulse of urea. By introducing a pulse of urea solution
of volume 200 ± 83 picoliters, we created a transient environ-
ment with a uniform urea concentration that is, at most, the con-
centration of the urea solution in the micropipette. Using this
assay, we empirically determined that H. pylori’s threshold of
urea detection in our assay is at least 50 nM (Figures 4B and
4C and Movie S5).

Urease Maintains Low Urea Concentrations around
H. pylori and Allows TlpB to Detect Low Concentrations
of Urea
The high affinity of TlpB for urea is difficult to reconcile with the
1–5 mM concentration of urea typically encountered in the stom-
ach (Blusiewicz et al., 2005), which would saturate the receptor.
However, H. pylori possesses a urease that rapidly destroys urea
and will effectively lower the concentration of urea near the bac-
teria. We therefore asked whether urease has a role in H. pylori’s
ability to sense urea and how it relates to the unusually high
affinity of TlpB for urea. We generated a DureAB mutant, which
lacks functional urease, and tested it for its ability to respond
to urea at concentrations ranging from 25 nM to 1 M in the micro-
gradient assay. We discovered that this mutant is unable to
respond to urea (Figure 5A). We also found that urea concentra-
tions greater than 500 mM inhibited WT H. pylori from respond-
ing (Figure 5B). High concentrations of urea have also been
shown to inhibit a chemotactic response in the capillary tube
assay (Mizote et al., 1997). We infer that such high concentra-
tions saturate both the receptor and urease, thereby preventing
the bacteria from detecting a gradient. These data suggest that
H. pylori’s own urease activity lowers the effective environmental
concentration of urea experienced by H. pylori to a range in
which the ultrasensitive TlpB receptor can function.
Indeed, addition of exogenous Jack Bean urease to a DureAB
culture restores the mutant’s chemotactic response to urea (Fig-
ure 5C and Movie S6). As exogenous urease is unlikely to reach
the periplasm of H. pylori, these data suggest that urease is not
directly involved in sensing urea. Rather, urease enhances
H. pylori’s sensitivity to urea by generating and maintaining a
steep gradient, thereby allowing TlpB to continually sense min-
ute concentrations of this host metabolite.
DISCUSSION
Our study has revealed that H. pylori is rapidly and sensitively
attracted to host metabolites that emanate from gastric organoid
epithelial cultures and intact polarized epithelial monolayers. We
uncovered the identity of one receptor responsible for recog-
152 Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc.
Figure 4. H. pylori’s High-Affinity Chemore-
ceptor TlpB Detects Nanomolar Amounts
of Urea
(A) Dose response curve of WT H. pylori to varying
concentrations of urea in the micropipette (10 mM–
1 mM). Each point represents the bacterial density
(pixel density) within a 60 mm radius from the
micropipette tip at 15 s post-injection. Blue line is a
log-log fit to the points.
(B) Motility tracings of WT versus DtlpB H. pylori’s
response at 13 s after the initiation of a 50 nM urea
pulse.
(C) Quantification of the responses of WT H. pylori
versus DtlpB to a pulse of 50 nM urea. Each point
represents the bacterial density (pixel density) at a
particular time in the recorded movie. Zero second
is defined as the moment the pulse was initiated
(n = 3 movies per condition). p value for scatter plot
indicates significance of time by group interaction
via a two-way repeated-measures ANOVA. See
also Movie S5.
nizing these metabolites as TlpB. We then identified one chemo-
attractant as urea, a ubiquitous byproduct of host metabolism
that diffuses across the epithelium and into gastric secretions.
By exploring the mechanism involved in the sensing of urea,
we were able to link this process of chemosensation to the ability
of H. pylori to alter its own microenvironment through the rapid
destruction of urea.
Previous researchers have speculated that urea emanating
from epithelial surfaces may attract H. pylori. This was based
on the fact that H. pylori was observed to be attracted to urea
through a capillary tube assay (Mizote et al., 1997; Nakamura
et al., 1998). Our results support these insights and also show
how H. pylori fine-tunes its ability to sense urea in the presence
of a powerful urease. Worku and colleagues reported that
H. pylori is attracted to human plasma (Worku et al., 2004).
Indeed, it is possible that serum in the capillary circulation under-
lying the gastric mucosa is one source of urea that diffuses
across the epithelium into the gastric lumen. This could explain
why damaged epithelium during ulcer formation attracts
H. pylori (Aihara et al., 2014). We show here that urea is also pro-
duced directly by cells that make up human gastric organoids
and that TlpB is sensitive enough to detect even the minute
amounts of urea that diffuse from epithelial monolayers.
Like commensal microbes that colonize the host over long
periods of time, H. pylori must sense and integrate many signals
in order to colonize and persist. Given the precarious nature of
living in the stomach, we predict that there are multiple signals
that are important to arrive at and persist in this delicate niche.
While our in vitro finding suggests that urea is the dominant
chemoattractant emanating from cells, there are likely other
host metabolites that attract H. pylori that are difficult to detect
with our assay if they are present in high concentrations in the
bacterial culture medium. In fact, cholesterol has been reported
to also attract H. pylori (Wunder et al., 2006). It will be valuable to
identify other host metabolites that direct H. pylori to the gastric
epithelium.
When we tested DtlpB in a murine model of infection, we did
not see a defect in the levels of initial colonization after 2 weeks
of infection. Since finding the gastric epithelium is essential for

(C) The responses of WT H. pylori versus DureAB in the presence or absence of exogenous Jack Bean urease to a microgradient formed by injecting 1 mM urea
are plotted as in (A) (n = 2 movies for WT and 3 movies for DureAB+/ exogenous urease).
p values for scatter plots indicate significance of time by group interaction via a two-way repeated-measures ANOVA. See also Movie S6.
H. pylori’s survival in the stomach, it is likely that there are other
host signals that H. pylori detects through its other chemorecep-
tors to find the epithelial surface and the protective mucus layer
of the stomach. Indeed, H. pylori’s TlpA, TlpC, and TlpD chemo-
receptors have been demonstrated to be important for coloniza-
tion of the stomach (Andermann et al., 2002; Rolig et al., 2012).
Thus, there may be compensatory signals sensed through the
other chemoreceptors that allow DtlpB to colonize the epithe-
lium. In future work we propose testing multiple combinations
of chemoreceptor mutants to better characterize the critical sig-
nals involved in establishing colonization of the stomach.
After 6 weeks of infection, however, we found that DtlpB does
have a defect in colonization compared to WT. These results
suggest that sensing through TlpB is important for persistence
in the stomach. This may be because H. pylori must continue
to sense urea to avoid the constant mechanical clearance mech-
anisms of the stomach or that it is important in avoiding clear-
ance by the immune response. Others did not find a significant
defect in DtlpB infection up to 6 months but did show a trend
in decreasing counts (Williams et al., 2007). The different results
may be due to differences in the experimental strains of H. pylori
or mice used or a secondary mutation in our DtlpB mutant. Un-
fortunately, we have been unable to generate a complemented
strain in PMSS1, but two independent DtlpB clones showed
similar results. While further studies are needed to determine
the role of TlpB in long-term persistence, no human clinical iso-
lates have been described to lack the tlpB gene, so it seems
likely that its activity in the human stomach is important. Interest-
ingly, it was recently reported that a small RNA modulates TlpB
expression in different strains of H. pylori (Pernitzsch et al., 2014).
The regulation of the gene expression of chemoreceptors will
also be important to investigate in the future.
Host-derived molecules have been demonstrated to be impor-
tant for bacterial colonization in other animal models. Notably, in
the symbiosis of the Hawaiian bobtail squid, Euprymna sco-
lopes, and the bioluminescent bacterium, Vibrio fischeri, a host
chitobiose gradient has been shown to attract V. fischeri to its
colonization site: the squid light organ (Kremer et al., 2013; Man-
del et al., 2012). Salmonella Typhimurium is known to induce
inflammation in the intestine and employs strategies to increase
its growth in this environment, which ultimately promotes its
Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc. 153
Figure 5. Urease Degradation of Urea Facil-
itates Urea Sensing through TlpB
(A) The responses of WT H. pylori versus DureAB
to 1 mM urea. Each point represents the bac-
terial density (pixel density) at a particular time
in the recorded movie. Zero second is defined
as the moment the micropipette was introduced
into the bacterial culture and a gradient was
initiated (n = 2 movies for WT and 3 movies for
DureAB).
(B) Dose response curve of WT H. pylori to varying
concentrations of urea in the micropipette (1 mM–
1 M). Each point represents the bacterial density
(pixel density) within a 60 mm radius from the
micropipette tip at 15 s post-injection. Blue line is
a log-log fit to the points.
transmission. Recently, it was shown that S. Typhimurium
thrives in the inflamed gut by chemotaxing toward nitrate and
tetrathionate, host byproducts produced as a result of the in-
flammatory response (Rivera-Chávez et al., 2013). Furthermore,
it was essential that S. Typhimurium utilize these compounds for
respiration in order to benefit from the inflamed gut in vivo.
Like Salmonella, H. pylori not only colonizes a specific niche in
the host, but it also modifies its host environment. It produces
chronic inflammation, alters epithelial biology, and with urease,
alters the concentration of urea and pH of the stomach in the
areas that it colonizes. While exploring the mechanism that un-
derlies ureataxis in H. pylori we found an important link between
the chemosensation of urea and the role of urease in modifying
the microenvironment. We show that H. pylori’s urea sensing is
intimately tied to the simultaneous destruction of this metabolite
by its urease enzyme. Urease is an essential virulence factor that
allows H. pylori to survive in the stomach presumably by
enhancing its ability to withstand transient exposures to hydro-
chloric acid through the buffering capacity of ammonia and bi-
carbonate (Eaton et al., 1991; Tsuda et al., 1994). We propose
that to benefit from the acid-buffering properties conferred by
urea degradation, H. pylori has evolved a chemoreception
mechanism for urea with exquisite sensitivity, involving a chemo-
receptor-ligand interaction of high affinity that functions at nano-
molar concentrations created locally by urease. The high affinity
of TlpB for urea and the efficient degradation of urea by urease
allow H. pylori to detect concentrations of urea as low as
50 nM. This permits H. pylori to continually sense urea emanating
from the gastric epithelium and direct its movement toward this
favored niche even when minute concentrations are present. We
thus describe a mechanism by which a bacterium navigates and
modifies the host environment through simultaneously sensing
and destroying a host metabolite.
EXPERIMENTAL PROCEDURES
Bacterial Strains and Culture Conditions
The previously published H. pylori strain PMSS1 (Arnold et al., 2011) was
used for all experiments in this study except those noted. H. pylori strains
were grown either on Columbia blood agar plates or in Brucella broth
supplemented with 10% fetal bovine serum at 37 C, 10% CO2, as described
previously (Amieva et al., 2002). Please see Supplemental Information for a
detailed description of how bacterial strains were generated, verified, and
cultured.
Human Gastric Organoid 3D Culture
Stomach specimens used in this study were obtained from patients that un-
derwent gastrectomy or gastric bypass surgery at Stanford Hospital. The
use of these specimens was approved by the Stanford University IRB. Spec-
imens received came from discarded tissue from surgical procedures and
were verified to be normal tissue by a pathologist. Isolation of gastric glands
was performed as previously described with minor modifications (Barker
et al., 2010; Sato et al., 2011). Please see Supplemental Information for a
detailed description of how organoids were cultured.
Test Solutions for Microgradient Assay
Organoid-conditioned media was obtained by incubating 250 ml DMEM
(GIBCO) on organoids for 10 hr after washing the Matrigel-embedded orga-
noid cultures 10 times with 500 ml DMEM. Caco-2 cell-conditioned media
was obtained by incubating 500 ml DMEM (GIBCO) on polarized monolayers
for 14 hr after washing the monolayer 5 times with 500 ml DMEM. MDCK
cell-conditioned media was obtained by incubating 500 ml DMEM (GIBCO)
on polarized monolayers for 7 hr after washing the monolayer 5 times with
500 ml DMEM. Urea solutions were made by dissolving ultra-pure urea into
sterile, distilled, and deionized water. For urease treatment experiments,
20 ml of a urease solution (0.1 g of Jack Bean urease [Fisher Scientific] resus-
pended in 500 ml of PBS) was added to the test solution and allowed to
incubate for 2 hr at 37 C and 10% CO2. Urease-treated organoid- or cell-
conditioned media was filtered through a 0.22 micron filter (Millipore) before
loading into the micropipette to prevent blockage of the micropipette by large
particulates.
Microgradient Assay
H. pylori cultures used for the assay were made by subculturing from a 16 hr
overnight culture and grown to an OD600 of 0.3. 270 microliters of the subcul-
ture was placed into the center of a glass-bottom 35-mm dish (MatTek) con-
tained by a ring of vacuum grease (Figure S1A). The dish was placed above
a 323 objective of a Zeiss Axiovert-35 inverted microscope equipped with
phase-contrast optics and a heated stage (37 C). A Hammamatsu C2400
video charge-coupled-device (CCD) camera was used to record via an
Argus-20 image processor onto Quicktime at 30 frames per second. A Femto-
tip II microinjection micropipette (Eppendorf) containing 8 ml of the test solution
was inserted into or removed from the viewing field using a micromanipulator
(Eppendorf 5171) (Howitt et al., 2011). To create a microgradient, a compensa-
tion pressure of 30 hPa was applied via the Eppendorf transjector 5246 to
maintain a constant flow at 0.372 picoliters/minute from the tip. This pressure
was determined empirically and selected because it did not physically affect
the bacteria swimming near the micropipette tip while generating a stable
gradient within the viewing field. For the urea pulse experiment, the contents
in the micropipette were released by holding down the clean function of
the transjector until the compensation pressure reached approximately
3,500 hPa. After 3,500 hPa was reached, the clean function was released
and the pulse ceased. We estimated that a pulse releases a volume of 200 ±
83 picoliters into the bacterial culture by measuring the change in pH of a water
sample after pulsing in 2.85 M HCl. The concentration of the pulsed solution
experienced by the bacteria is at most the concentration of the solution in
the micropipette.
Quantification of Bacterial Chemotactic Responses to
Microgradients
H. pylori chemotactic responses were quantified from phase contrast video
microscopy movies recorded at 30 frames per second using ImageJ
software version 1.46r. Movies were analyzed starting 4 s before micropi-
pette insertion (pre-injection) to 20 or 30 s after micropipette insertion
(post-injection). After background subtraction and contrast adjustment,
0.5 s segments of the movies (15 frames) were combined into Z-projections
to generate traces of moving bacteria (Figure S1B). The frames containing
motility tracings were then thresholded into binary images. A circle with
a radius of 400 pixels (approximately 60 microns) centered at the micropi-
pette tip was drawn as the region of interest (ROI). The number of black
154 Cell Host & Microbe 18, 147–156, August 12, 2015 ª2015 Elsevier Inc.
pixels (proxy for bacteria) normalized to the area of the ROI was quantified
for each image and represented as pixel density at that time point. Re-
sponses are shown as either scatter plots, which display the pixel densities
from 4 s pre-injection to 20 or 30 s post-injection, or bar graphs, which
display the pixel density at 4 s pre-injection and 15 s post-injection. Statisti-
cal significance between the number of black pixels pre-injection versus
number of black pixels post-injection in the bar graphs or of the overall
response in the scatter plots was assessed using a two-way repeated-
measures ANOVA.
Animal Experiments
All animal experiments were performed in accordance with NIH guidelines and
with approval from the Institutional Animal Care and Use Committee of Stan-
ford University. 6-week-old female C57BL/6J mice were purchased from The
Jackson Laboratory. Animals were infected intraorally by allowing the animals
to drink a 5 ml suspension containing 108 CFU of H. pylori grown in Brucella
broth from a pipette tip (Howitt et al., 2011). Animals were sacrificed at 2, 4,
or 6 weeks post-infection. The stomach was harvested with the forestomach
removed and discarded, opened via the lesser curvature, and laid flat onto fil-
ter paper. Luminal content was removed, and the stomach was divided into
halves that spanned the corpus to the antrum. One half of the stomach was
weighed and mechanically homogenized for 30 s in 1 ml Brucella broth.
Homogenized stomachs were serially diluted and plated for CFU counts.
The data represent the number of CFU/gram of stomach. Statistical signifi-
cance in the recovered bacterial load between WT and mutant was assessed
by a Mann-Whitney test.
Analysis of TpB Ligand Binding Configurations
Diffraction data and models as previously described (Goers Sweeney et al.,
2012) were downloaded from the RCSB Protein Data Bank (Berman et al.,
2000). Discussed include TlpB bound to urea, hydroxyurea, acetamide, and
formamide (PDB: 3UB6, 3UB9, 3UB7, and 3UB8, respectively). ‘‘Omit’’ differ-
ence electron density maps were inspected, and minor adjustments to the
atomic models were made and refined using the Coot and CCP4 program
suites (Emsley et al., 2010; Winn et al., 2011). In particular, hydroxyurea was
determined to bind as a mixture of the E- and Z-isomeric configurations,
each in a different orientation, with roughly equal occupancy. Potential
protein-ligand hydrogen bonds were evaluated according to criteria set forth
by Baker and Hubbard (Baker and Hubbard, 1984).
Thermal Stability Assay—Thermofluor Assay
All data were collected on an Applied Biosystems StepOnePlus machine.
Samples were pipetted into wells of a 384-well plate and covered with optically
clear plastic prior to data acquisition. Each well contained 20 ml of total volume
in water that included 136 mM NaCl, 0.21 mg/ml TlpBpp protein (periplasmic
region only), 14.53 SYPRO Orange (Invitrogen), 57 mM Bis-Tris, 25 mM citric
acid (pH 5), and urea or urea analog to the final concentration needed
(0–50 mM). Thermal denaturation runs consisted of a fast ramp from 25 C to
4 C (hold for 2 min) and a slow ramp from 4 C to 80 C (hold). During the
slow ramp, about 2.5 fluorescence measurements were taken per each
increase in 1 C. As the temperature increases and the protein melts, the
fluorescence increases to a maximum value when the protein is fully melted
(producing a melting curve). The melting temperatures (Tm) were approxi-
mated by taking the minimum fluorescence value prior to the fluorescence
peak and the maximum fluorescence value and then taking the mid-point
(inflection point).
Statistical Analyses
All microgradient assay data comparing pixel density at 4 s pre-injection to
pixel density at 15 s post-injection, as well as the scatter plots of pixel densities
over time, were analyzed via a two-way repeated-measures ANOVA test in the
GraphPad Prism 6 software program. The p value for the scatter plots indi-
cates the significance of the time by group interaction via two-way
repeated-measures ANOVA. For animal experiments, statistical significance
was assessed via a Mann-Whitney test. Center values are means, and error
bars represent SD. n indicates the number of movies per condition or the
number of animals used. NS indicates no statistical significance, *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and six movies and can be found with this article online at
http://dx.doi.org/10.1016/j.chom.2015.07.002.
AUTHOR CONTRIBUTIONS
J.Y.H. and M.R.A. conceived the study. J.Y.H., E.G.S., K.G., and M.R.A. de-
signed the experiments. J.Y.H. performed most of the experiments. E.G.S
and S.J.R. provided the thermofluor assay data and analysis of ligand binding
to the TlpB periplasmic domain crystal structure. H.Z., M.A.C., C.J.K., and
M.S. optimized conditions to grow the human gastric organoids and generated
the organoids for this study. J.Y.H., E.G.S., K.G., and M.R.A. analyzed the data
and wrote the manuscript. All authors provided comments on the manuscript.
ACKNOWLEDGMENTS
We thank members of the Amieva lab for comments on the manuscript. We
also thank Karen Ottemann for providing us the antibody against the chemo-
receptors. This research was supported by the DoD Air Force Office of
Scientific Research NDSEG Fellowship and the Stanford Diversifying
Academia Recruiting Excellence Doctoral Fellowship (to J.Y.H.); the Medical
Research Foundation Oregon Scientist Development Award (to E.G.S.); the
German Research Foundation DFG, Si 1983/1-1 (to M.S.); the R. Robert &
Sally Funderburg Research Award in Gastric Cancer and the Morgridge
Faculty Scholar Award (to M.R.A.); R01DK101314 (to M.R.A., K.G., and
S.J.R.); U19AI116484-01 (to M.R.A. and C.J.K.); and NIH 2U01DK085527,
1U01CA151920, and 1U01CA168424 (to C.J.K.).
Received: March 20, 2015
Revised: June 8, 2015
Accepted: July 7, 2015
Published: August 12, 2015
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