Mucosal penetration primes Vibrio cholerae for host
colonization by repressing quorum sensing
Zhi Liu*, Tim Miyashiro†, Amy Tsou*, Ansel Hsiao*, Mark Goulian†‡, and Jun Zhu*§
Departments of *Microbiology,†Physics, and‡Biology, University of Pennsylvania, Philadelphia, PA 19104
Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved April 17, 2008 (received for review March 7, 2008)
To successfully infect a host and cause the diarrheal disease
cholera, Vibrio cholerae must penetrate the intestinal mucosal
layer and express virulence genes. Previous studies have demon-
strated that the transcriptional regulator HapR, which is part of the
quorum sensing network in V. cholerae, represses the expression
of virulence genes. Here, we show that hapR expression is also
modulated by the regulatory network that governs flagellar as-
sembly. Specifically, FliA, which is the alternative ?-factor (?28)
that activates late-class flagellin genes in V. cholerae, represses
hapR expression. In addition, we show that mucin penetration by
V. cholerae is sufficient to break flagella and so cause the secretion
of FlgM, the anti-? factor that inhibits FliA activity. During initial
colonization of host intestinal tissue, hapR expression is repressed
because of low cell density. However, full repression of hapR
expression does not occur in fliA mutants, which results in atten-
uated colonization. Our results suggest that V. cholerae uses
flagellar machinery to sense particular intestinal signals before
colonization and enhance the expression of virulence genes by
modulating the output of quorum sensing signaling.
is still endemic in many developing countries (1). To survive in
to changing environmental signals (2). One such signal is the
local V. cholerae cell density, which is sensed through a process
known as quorum sensing (3). V. cholerae measures its popula-
tion density by producing, secreting, and monitoring the con-
centration of at least two autoinducers (4). The sensory infor-
mation provided by autoinducer concentration is channeled
through the response regulator LuxO. In its phosphorylated
state, LuxO represses hapR transcription by activating the ex-
pression of several small regulatory RNAs (5, 6). At high cell
density, LuxO is not phosphorylated, so production of HapR
increases. HapR controls a number of cellular functions and
indirectly regulates the expression of multiple virulence genes (6,
7). As V. cholerae colonizes the small intestine and multiplies, it
activates a cascade of regulatory proteins that leads to the
production of an array of virulence factors [supporting infor-
mation (SI) Fig. S1]. The membrane-localized ToxRS and
TcpPH regulatory complexes respond to host environmental
signals to initiate this cascade, which culminates in the produc-
tion of ToxT, which directly up-regulates virulence genes en-
coding Cholera Toxin and the Toxin Coregulated Pilus (TCP)
(8). Expression of tcpPH requires the transcriptional regulator
AphA (9). Quorum sensing and pathogenesis are coupled
through the action of HapR, which represses the transcription of
aphA and thus inhibits optimal virulence factor production (10)
(see Fig. S1). Because high cell densities are common during the
late stage of infection, HapR-mediated repression of coloniza-
tion and virulence genes is thought to help V. cholerae to detach
to find a new site of infection or exit the host and initiate a new
infectious cycle (7, 11). Although the action of HapR links
quorum sensing and virulence gene regulation, the pathways by
which quorum sensing is regulated in the host and the exact roles
played by quorum sensing during V. cholerae infection have not
he Gram-negative bacterium Vibrio cholerae is the causative
agent of cholera, an acute dehydrating diarrheal disease that
In addition to the production of virulence factors, for V.
cholerae to colonize the villus epithelial cells within the small
intestine, bacteria must swim through a protective mucus gly-
cocalyx (12). It has been proposed that the motility conferred on
V. cholerae by its single polar flagellum is necessary for this
process (12, 13). Flagellar biogenesis is complex and involves a
combination of transcriptional, translational, and posttransla-
tional regulation (14). Flagellar biosynthesis genes can be cate-
gorized into three classes (early, middle, and late) based on their
order of activation. In V. cholerae, FlrA and the ?54-holoenzyme
transcribe early genes, including those that encode the Motor/
Switch ring and export components. The middle genes, encoding
structural and assembly proteins that form the hook-basal-body
(HBB) of the flagellum, are activated by FlrC and the ?54-
holoenzyme. After formation of the HBB, the anti-?28factor
(FlgM) is secreted from the cell, allowing ?28(FliA) to activate
transcription of late genes, which encode the flagellin proteins
and motor components (15, 16).
In this study, we identify components of flagellar biosynthesis
that also control quorum sensing via regulation of hapR expres-
sion, suggesting a link between regulation of motility and
regulation of quorum sensing in V. cholerae. This combination
efficiently prepares bacteria for accessing colonization sites and,
at the same time, allows for the maximal production of virulence
Identification of Additional Regulatory Factors Involved in Quorum
Sensing. To identify factors that regulate virulence genes through
the quorum sensing pathway in V. cholerae, we designed an
antibiotic-coupled transposon screen. We fused the zeocin-
resistance gene sh ble (17) to the ToxT-activated promoter of the
TCP subunit gene tcpA and integrated the resulting cassette into
the lacZ locus of wild-type, luxO and hapR strains. Wild-type and
hapR strains containing this cassette are resistant to zeocin after
growth in AKI medium, which induces the expression of viru-
lence genes (18). However, the corresponding luxO mutant is
sensitive to zeocin when grown in AKI medium (Table S1). The
latter result is consistent with low-virulence gene expression
caused by the high levels of HapR in a luxO mutant (7, 10) (Fig.
S1). We performed transposon mutagenesis in this luxO
lacZ::PtcpA-sh ble strain (LZV8) using the mariner transposon
TnAraOut (pNJ17), which contains araC and a PBADpromoter
that transcribes away from the transposon (19). The addition of
arabinose derepresses the PBAD promoter by removing AraC
We reasoned that if the transposon disrupts a gene required for
and J.Z. analyzed data; and Z.L., T.M., A.H., and J.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
July 15, 2008 ?
vol. 105 ?
no. 28 ?
and PtcpA-sh ble will be induced and confer zeocin resistance.
Similarly, if the transposon inserts into the promoter region of
a gene that either represses hapR expression or bypasses the
HapR repression of aphA, then zeocin resistance will arise in the
presence of arabinose. To distinguish between mutations that
affect HapR directly and those that affect regulation down-
stream of HapR, we included the Vibrio harveyi luxCDABE
reporter construct, which is activated directly by HapR (6).
From 10 transposon libraries each containing ?109cells, we
obtained ?500 zeocin-resistant colonies. Table S1 summarizes
the locations of the transposon insertions and their related
phenotypes. More than 50% of these mutants have transposons
inserted in the hapR locus and exhibited the rugose-colony
phenotype associated with a mutation in hapR (20, 21). Because
disruption of hapR relieves the repression of the aphA promoter
by HapR, strains containing transposon insertions within hapR
are zeocin-resistant. Many transposon insertions occurred up-
stream of the PtcpA-sh ble construct resulting in overexpression of
the zeocin-resistance gene in the presence of arabinose. We
that are resistant to zeocin in the presence of arabinose, further
confirming that HapR inhibits the expression of virulence genes
by repressing aphA. Disruption of the H-NS-like DNA-binding
protein VicH (VC1130) also resulted in zeocin resistance;
studies have shown that deletion of vicH results in high expres-
sion of the genes encoding cholera toxin, TCP, and ToxT (22).
However, the lux expression pattern of vicH mutants was the
same as that of the parental luxO mutant, indicating that the
increased tcpA expression was not due to a regulatory defect in
quorum sensing. Similarly, mutations that disrupt either VC2305
(ompK) or VC2271 (ribD) restore both cholera toxin and TCP
production in the luxO mutant without changing lux expression.
The mechanism that enables these two mutants to increase the
expression of virulence genes independent of quorum sensing is
the subject of another study.
Interestingly, we obtained a high number of mutants with
transposon insertions in genes involved in flagellar biosynthesis
(Table S1). Disruption of the flgBCD genes, which encode
flagellar rod proteins (23), activated PtcpA-sh ble in the luxO
mutant. We also obtained a number of arabinose-dependent,
zeocin-resistant mutants with transposon insertions located up-
stream of fliA, which encodes the ?-factor required for activation
of certain flagellin genes by RNA polymerase (RNAP) (16). All
of these flagellar mutants produce CT and TCP (Table S1). In
addition, luxO mutants that harbor such flagellar mutations
exhibit reduced lux expression (Table S1), suggesting that quo-
Deletion of flgD Enhances the Expression of Virulence Genes Through
of virulence genes, we constructed strains with an in-frame
deletion of flgD, which encodes one of the flagellar rod proteins.
As expected, flgD mutants are nonmotile ((Fig. S4A). Although
TcpA and CT production was abolished in the luxO mutant,
wild-type levels of TcpA and CT were detected in both flgD and
flgD luxO mutants (Fig. S2A). To determine whether the dele-
tion of flgD alters aphA expression in the luxO mutant, we
measured the corresponding activity levels of an aphA-lacZ
transcriptional reporter. As expected, deletion of luxO results in
repression of aphA (Fig. S2B). However, mutation of flgD not
only results in wild-type levels of aphA transcription but also
restores aphA expression in the luxO mutant. This explains why
the inhibition of the virulence regulon in the luxO mutant was
of flgD in quorum sensing, we transformed wild-type and flgD
strains with pBB1, which contains the luxCDABE operon from
V. harveyi that can be regulated directly by HapR (6). Although
lux expression depends on cell density in both cases, overall
expression is lower in the flgD strain than in the wild-type strain
(Fig. S2C). We attribute this reduction in lux expression to lower
levels of HapR because the expression of a hapR-lacZ transcrip-
tional reporter was also inhibited in the flgD mutant (Fig. S2D).
Taken together, these results indicate that flagellar synthesis
modulates the quorum sensing output, including the expression
of virulence factors, by regulating hapR transcription.
The Alternative ?-Factor FliA (?28) Represses hapR Expression. The
regulatory hierarchy that determines the order for expressing
flagellar genes in V. cholerae ends with the ?28-dependent
activation of particular flagellar genes (e.g., flaBCD) (16).
Because constitutive expression of fliA, which encodes ?28,
inhibits regulation by quorum sensing (Table S1), we hypothe-
sized that the repression of hapR observed in flgB, flgC, and flgD
of FliA increases upon secretion of the anti-?28factor FlgM
through the flagellar export apparatus (15, 16). To determine
whether this process plays a role in the altered hapR regulation
of the flgD mutant, we compared the level of FlgM in superna-
tants of wild-type and flgD cultures. The level of FlgM present
in the supernatant of the flgD mutant is higher than that of
wild-type V. cholerae (Fig. 1B), suggesting that disrupting flagel-
lar rod assembly leads to higher FliA activity by increasing FlgM
secretion. Consistent with this hypothesis, the expression of the
FliA-activated gene flaD was enhanced in a flgD mutant (Fig.
S3). To further investigate the relationship between FliA–FlgM
for repression of the hapR promoter by secretion of FlgM. (B) FlgM secretion
is increased in the flgD mutant. Strains containing a plasmid expressing a
functional flgM-his6 were grown to midlog. Culture pellets (P) and TCA-
precipitated supernatants (S) were isolated and subjected to Western blot
analysis using anti-His-6 antiserum. All samples were normalized to contain
109bacterial cells. (C) Reduced production of HapR is due to hapR repression
by FliA. Strains harboring hapR-lacZ transcriptional fusions were grown to
midlog in LB and harvested to measure ?-galactosidase activity (Upper).
Results, reported in Miller units, are means of three experiments ? standard
deviations. Whole-cell extracts were subjected to Western blot analysis using
anti-HapR antiserum (Lower). All samples were normalized to contain 109
FliA represses quorum sensing in flgD mutants. (A) Proposed model
www.pnas.org?cgi?doi?10.1073?pnas.0802241105 Liu et al.
interaction and hapR expression, we measured hapR transcrip-
tion and HapR production in various mutants grown to midlog
phase. Whereas hapR expression is inhibited in the flgM and flgD
mutants, hapR is overexpressed in the fliA mutant (Fig. 1C).
More importantly, deletion of fliA in the flgD mutant restores
HapR production, indicating that the effect of hapR repression
in flgD mutants is mediated through FliA. Furthermore, a fliA
mutant complemented by a plasmid that constitutively expresses
fliA restores the repression of hapR. Mutations in flaD and motY,
which are known to be regulated by FliA (16), did not affect
FlgM secretion or hapR expression (data not shown). Taken
together, these results suggest that the high activity of FliA in
either flgM or flgD mutants inhibits hapR expression. At this
time, we do not know whether FliA regulates hapR directly or
through another regulatory mechanism. The alternative ?-factor
?28could activate the expression of unknown repressor to
repress the hapR expression or activate small RNAs to regulate
hapR mRNA. In fact, hapR has been shown to be regulated by
RNA-binding protein Hfq and a set of sRNAs activated by LuxO
and ?54(5). However, if sRNAs are involved in ?28-mediated
hapR repression, they are different from those sRNAs induced
by LuxO because ?28represses hapR in a luxO mutant (Fig. S2).
Furthermore, because ?28also represses hapR in an hfq mutant,
this regulation is independent of Hfq (data not shown).
Repression of Quorum Sensing by FliA Is Important for Proper Intes-
tinal Colonization. Flagella are thought to help V. cholerae swim
through mucosal layers to colonize the intestinal surface (13).
Consistent with this hypothesis, flgD, fliA, and flgM mutants,
which have reduced motility (15) (Fig. S4A), do not colonize the
intestines of infant mice as well as motile wild-type cells (Fig.
2A). The observation that a fliA mutant displays a severe
colonization defect was intriguing because FliA represses hapR
expression (see Fig. 1). Colonization efficiency was significantly
increased in a fliA hapR mutant (Fig. 2A) as compared with the
fliA single mutant (P value ?0.01). Consistent with previous
reports (7), the hapR mutant colonized mice as well as wild-type.
In addition, an in vivo colonization competition assay with a fliA
single mutant and a fliA hapR double mutant demonstrated that
the double-mutant colonizes ?6-fold better than the fliA mutant
(Fig. S4B). These results suggest that the colonization defect of
the fliA mutant is partially due to insufficient repression of hapR.
To provide further in vivo evidence that hapR expression is
higher in fliA mutants, we colonized infant mice using strains
that contain a hapR-Kmrtranscriptional fusion, which confers
kanamycin resistance to cells expressing hapR (24). At 4 h, hapR
expression in wild-type cells was low, presumably because of the
small number of cells that had colonized the small intestine (Fig.
2B). However, by 18 h, the number of cells in the small intestine
had increased, and hapR expression was high. The hapR expres-
sion patterns of flgD, fliA, and flgM mutants in vivo were similar
to those in vitro (compare Figs. 1C and 2B), with significantly
2B). Taken together, these results suggest that the repression of
hapR by FliA is required for proper colonization of host
V. cholerae Loses Its Flagellum and Represses Regulation by Quorum
Sensing During Mucosal Penetration. The results described above
strongly suggest a link between the regulatory networks of
quorum sensing and flagellar assembly during intestinal coloni-
zation by V. cholerae. We hypothesized that the cross-regulation
may occur while colonizing bacteria cross the mucosal layer of
the intestinal surface, because flagella are thought to be impor-
tant for mucus penetration. To approximate this stage of infec-
tion, we used an in vitro assay based on the migration of cells
through a column of mucin (25, 26). Wild-type cells migrated
through the mucin column significantly faster than nonmotile
In addition, antibiotic-killed bacteria failed to penetrate the
mucin column (data not shown). These data suggest that flagella
may help V. cholerae swim through mucosal layers. Surprisingly,
both flagellar staining and electron microscopy revealed that the
majority of wild-type cells (?80%) had lost their flagella while
migrating through a column containing 1% mucin (Fig. 3B). It
should be noted that the absence of flagellar structures observed
in these experiments is not due to the interference of mucin with
flagellar staining or microscopy, because flagellar structures are
apparent when mucin and bacteria are mixed and then stained
for flagella (data not shown). To determine whether bacteria are
motile after penetrating mucin, we deposited a suspension of
cells onto a transwell containing a thin layer of mucin on top of
a 3-?m filter and monitored the motility of the cells that passed
through the filter. Consistent with our electron microscopy
of infant mice. (A) In vivo competition assays. Infant CD-1 mice were orally
inoculated with ?106wild-type and mutant bacteria. For each mutant, the
competitive index is defined as the number of colony-forming units (CFU) for
the mutant compared with the corresponding CFU number for a wild-type
strain recovered from the intestines 18 h after inoculation. (B) Expression of
hapR during colonization. Infant CD-1 mice were orally inoculated with ?106
bacteria that contain a hapR-Kmrreporter. At each time point, mouse intes-
tines were homogenized, treated with or without kanamycin (500 ?g/ml) for
10 min, and plated on LB plates. The number of colonized bacteria per mouse
(above each bar) was calculated based on the number of CFU recovered from
samples not treated with kanamycin. Expression of hapR is defined as the
percentage of Km-resistant CFU of the total CFU. Results are means from
The expression of hapR is repressed by FliA during the colonization
Liu et al.
July 15, 2008 ?
vol. 105 ?
no. 28 ?
results, we found that bacteria that passed through the mucin
layer and reached the outer chamber were not motile (see
MovieS1 and MovieS2).
The above experiments indicate that V. cholerae cells use
flagella to penetrate mucin layers but lose them during the
process. Because we did not observe intact flagella on cells
within mucin, we hypothesized that V. cholerae uses an alterna-
tive mechanism to translocate through the mucin. To test this, we
compared the rates of migration through a 1% mucin column for
wild-type and flgD mutant cells that had been premixed with
mucin. Consistent with our hypothesis, we found that the cells
migrated through the mucin column at similar rates (Fig. 3A
are important for intestinal colonization only during the initial
penetration of mucin. The exact mechanism used by V. cholerae
to cross mucosal layers is currently unknown and may involve
flagellum-independent motility used by V. cholerae (27) and
other bacteria (28).
To determine whether the loss of flagella from the exposure
of cells to mucin leads to increased FlgM secretion, we measured
FlgM levels in the cytoplasmic and extracellular fractions of cells
grown in the presence of mucin. The level of FlgM in the
supernatant of cultures supplemented with mucin was higher
than that of cultures without mucin (Fig. 4A), suggesting that
FliA activity increases during interaction between V. cholerae
and mucin. Consistent with our observation of hapR repression
by FliA (Figs. 1C and 2B), we also found that hapR expression
is repressed when V. cholerae penetrates mucin (Fig. 4B).
repressed in a fliA mutant as well as in a fliF mutants, which is
transcription of the FliA-activated gene flaD increased when V.
cholerae penetrates mucin (Fig. 4B). Taken together, these
results suggest that exposure to mucin is sufficient to activate
FliA, which inhibits regulation of quorum sensing by repressing
To further test the in vivo effect of the repression of hapR by
exposure of cells to mucin, we turned to a cell-culture infection
with a layer of 1% mucin or left untreated. The expression of
hapR was lower in those HEp-2-bound V. cholerae that had to
pass through mucin to reach the epithelial cell monolayer (Fig.
4C). However, for strains overexpressing fliA, hapR expression
remained low whether or not mucin was present. Consistent with
the colonization assays and mucin-penetration assays described
mutants swim more slowly through mucin than do wild-type cells. Midlog
cultures (100 ?l) of wild-type V. cholerae, the flgD mutant (white bars), or
cultures premixed with 100 ?l of 1% mucin were loaded into a column
of V. cholerae during mucin column penetration. Flagellar staining (Upper)
and transmission electron microscopy (Lower) of V. cholerae cells in the
V. cholerae cells lose flagella during mucin penetration. (A) Flagellar
Secretion of FlgM increases during mucin penetration. Midlog cultures of
wild-type bacteria expressing flgM-his6 were incubated for 30 min in LB
medium with or without 1% mucin. Cytoplasmic and cell-free supernatant
fractions were subjected to Western blot analysis. All samples were normal-
ized to contain 108bacterial cells. (B) Real-time RT-PCR analysis of hapR
expression. Mucin penetration assays were performed as described above.
RNA was extracted, and real-time RT-PCR was performed for hapR and flaD
transcripts. Transcript levels were normalized by 16S RNA. Results for each
to the transcript level in the absence of mucin. (C) Expression of hapR in V.
cholerae cells on the surface of HEp-2 cells. Confluent HEp-2 cell monolayers
were submerged in 1 ml of 1% mucin or LB. Midlog cultures of wild-type (wt)
and fliA-overexpressing (fliAC) strains of V. cholerae that contain the hapR-
Kmrreporter were inoculated onto HEp-2 cell cultures. After 2-hr incubation,
the bacteria bound to HEp-2 cells were collected, treated with or without
kanamycin (500 ?g/ml) for 10 min, and plated onto LB agar. The hapR-Kmr
expression was defined as the percentage of CFU that survive treatment with
kanamycin of the total CFU. All results are means of three experiments ?
Quorum sensing is repressed when V. cholerae penetrate mucin. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0802241105 Liu et al.
above, these results indicate that regulation of quorum sensing
is repressed through ?28during mucosal penetration.
The facultative human pathogen V. cholerae must rapidly adapt
to different environments, such as from its natural aquatic
habitats to the human digestive system, during its life cycle. To
do this, V. cholerae uses complex signal transduction pathways
that modulate its gene expression in response to various envi-
ronmental cues. The quorum sensing regulatory network mon-
density; however, other components also regulate quorum sens-
ing by modulating transcription of the regulator HapR (29–31).
In this study, we discovered that flagellar components also
regulate hapR expression. Specifically, we show that hapR tran-
scription is repressed by fliA, which encodes the alternative
?-factor involved in flagellar synthesis (?28). Similarly, removal
of flagellar rod proteins, which results in high levels of active ?28
because of increased secretion of the anti-?28protein FlgM,
inhibits hapR transcription.
Why does V. cholerae integrate motility and quorum sensing
regulatory pathways? One possibility is to further derepress the
virulence genes that are repressed by quorum sensing. Unlike
some enteric pathogens, which successfully infect hosts with a
low starting dose [e.g., Shigella (32)], V. cholerae requires ?106
cells to induce symptomatic cholera (33). However, the number
of V. cholerae cells that reach the small intestine is reduced by the
hostile compounds found in host environments, such as gastric
acid and bile salts (2). This reduction in cell number decreases
the concentration of autoinducer, which, in conjunction with a
HapR negative-feedback loop (34), represses hapR transcrip-
tion. Our results suggest that this reduction in cell number may
not be sufficient to completely inhibit HapR-mediated repres-
sion of virulence gene expression.
At their sites of colonization at mucosa, V. cholerae must
penetrate the thick glycocalyx of mucins that cover and protect
the intestinal epithelium. However, in penetrating this barrier,
FlgM is secreted (Fig. 4). This leads to derepression of FliA and
further repression of hapR through the activation of FliA.
Through hapR repression, V. cholerae cells are primed for
intestinal colonization, which is an important step leading to the
onset of cholera. This model is illustrated in Fig. S5. Consistent
colonizes infant mice better than a fliA single mutant (Fig. 2).
Furthermore, the higher hapR expression (as compared with
mutant also supports this model. Interestingly, a recent report
suggests that Salmonella can sense the wetness of its surrounding
environment by controlling the amount of FlgM secreted
through its flagellar apparatus (35). Similarly, our model implies
that V. cholerae uses flagella to sense host mucosal signals to
regulate genes that allow the cell to adapt to host intestinal
Previous studies suggest that the ability of the current pan-
demic V. cholerae strains to swim into the mucosal layer is
important for colonizing the intestinal surface (13, 36). Consis-
tent with these reports, we found that nonmotile strains colonize
10–25 times less efficiently than wild-type strains (Fig. 2A).
Furthermore, using mucin column-penetration assays, we found
that nonmotile strains penetrate mucin layers more slowly than
wild-type V. cholerae (Fig. 3A). However, many V. cholerae cells
lose their flagella while penetrating mucin layers (Fig. 3B),
suggesting that intact flagella are not required for the cells to
migrate through mucin once they have passed the initial inter-
through mucin, but once flagella are lost in this process, other
processes assist in the passage of bacteria through the glycocalyx
with mucin, they migrated as fast as wild-type cells (Fig. 3A
Right). Another possibility is that bacteria continuously regen-
erate flagella to replace those lost while penetrating mucus
layers. In fact, cells that encounter mucin secrete FlgM and so
begin to express FliA-regulated genes such as flaD (Fig. 4 A and
B). However, a recent study reported that FlgM/?28-dependent
gene expression in Salmonella enterica remains unchanged by
flagellar shearing despite the rapid regeneration of flagella (37).
Whether the cellular response to mechanically sheared flagella
is similar to that of breaking flagella during mucus penetration
is not clear.
The ability of V. cholerae to colonize and cause disease
requires tight control over the expression of multiple virulence
factors. However, pathogenesis and the associated genetic reg-
ulatory events in the host are not a series of disconnected
cascades nor do they depend only on the activation of virulence
regulators. Instead, the complex infection cycle of this pathogen
depends on a variety of genetic regulatory strategies, including
the repression of genes that inhibit colonization. It has already
been shown that V. cholerae reciprocally regulates the transcrip-
tion and biogenesis of virulence determinants and that of the
type IV MSHA pili to evade host immune defenses (26, 38). In
this study, we show that another reciprocal regulatory mecha-
nism serves to allow this pathogen to maximize virulence during
the mucosal barrier protecting the cells of the small-intestinal
epithelium. In the process, the flagella are lost, which leads to
repression of hapR, which encodes a negative regulator of
virulence genes. Thus, V. cholerae is capable of ‘‘dual use’’ of
necessary virulence processes such as flagellar motility and TCP
biogenesis to position and equip itself for colonization while
simultaneously inhibiting anticolonization factors such as HapR
and MSHA. This enables V. cholerae to access efficiently its
preferred colonization niches during the critical early phase of
infection, where a small number of bacterial cells must attach to
and establish themselves at the epithelium. It is this regulatory
flexibility that has made V. cholerae a potent human pathogen,
and a striking example of the complexities possible in bacterial
virulence regulation in disparate environments.
Strains, Plasmids, and Culture Conditions. All V. cholerae strains used in this
study were derived from E1 Tor C6706 (39). Strain and plasmid constructions
are described in SI Text.
Transposon Screen for hapR Repression. LZV8 (lacZ::tcpA-sh ble, ?luxO) was
mutagenized with the TnAraOut mariner transposon (19).The resulting li-
braries were inoculated into AKI medium containing 0.05% arabinose and
incubated without shaking at 37°C for 4 h until the OD600was ?0.1. Cultures
were treated with zeocin (25 ?g/ml) for 30 min at 37°C and plated on LB agar
without zeocin. Arbitrary PCR (19) and DNA sequencing were performed to
identify transposon insertion sites.
hapR Transcription Measurements. The hapR-lacZ transcriptional reporter was
integrated into the chromosomes of various flagellar mutants. ?-Galactosidase
assays were performed as described (7).
In the mouse colonization and mucin-penetration assays, hapR expression
was measured by using strains harboring the hapR-Kmrreporter, which is at
the hapR locus and maintains an intact copy of hapR (24). Bacteria isolated
from mouse intestines or mucin columns were added to fresh LB in the
This treatment is sufficient to kill 100% of V. cholerae cells that do not carry
LuxO (40). After treatment, the samples were plated onto LB. Expression of
hapR was defined as the number of kanamycin-resistant CFU normalized by
the number of total CFU.
For the mucin-penetration assays, hapR transcription was measured by
quantitative real-time PCR. Total RNA was isolated from column samples by
using TRIzol reagent (Invitrogen) and cleaned with the RNeasy kit (Qiagen).
RNA reverse transcription was performed by using the SuperScript II kit
Liu et al.
July 15, 2008 ?
vol. 105 ?
no. 28 ?
(Invitrogen) with 200 ng of RNA sample. Quantitative real-time PCR using Download full-text
primers specific for hapR was performed on an Opticon 2 system (MJ Re-
search). The 16S ribosomal RNA was used for an internal control in all
Infant Mouse Colonization Assay. The infant mouse colonization assay was
6-day-old CD-1 suckling mice. At different time points, intestines were ho-
mogenized, serially diluted, and plated onto LB agar. Bacterial numbers were
determined from the number of CFU.
Mucin-Penetration Assay and Flagella Visualization. Mucin columns were pre-
pared by adding different concentrations of bovine submaxillary mucin
(Sigma) to 1-ml syringes. Midlog bacterial cultures (100 ?l)or 100 ?l of cells
premixed with 1% mucin were loaded on the top of the mucin columns and
bottom of the mucin columns. Bacteria numbers were measured by serially
diluting samples, plating onto LB agar, and counting CFU. A drop of the
collected fractions was placed on slides to detect flagella by using the flagella
Pennsylvania Bioimaging Core).
his6 was introduced into various strains of V. cholerae. All samples were
bacterial cells. Supernatants were concentrated by using TCA precipitation
(10% trichloroacetic acid). Both extracellular and cytoplasmic proteins
were separated by SDS/PAGE, transferred to a nitrocellulose membrane,
and immunoblotted with affinity purified anti-6? His rabbit antiserum
epithelial cells were propagated in DMEM supplemented with 10% FBS (Sigma)
LB with or without 1% mucin. Midlog cultures of wild-type and fliA-
overexpressing strains of V. cholerae that contain the hapR-Kmrreporter were
37°C with 5% CO2, the medium was removed, and epithelial cells were washed
three times with PBS buffer to remove unbound bacteria. HEp-2 cells were lysed
the kanamycin treatment described above.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health (NIH) Grant R01AI072479 (to J.Z.), National Science Foundation Grant
(to T.M.) and a NIH T32 Bacteriology training grant (to A.T.).
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