Vibrio cholerae represses polysaccharide synthesis to promote motility in mucosa.

Vibrio cholerae Represses Polysaccharide Synthesis To Promote Motility in Mucosa Zhenyu Liu,a,b Yuning Wang,a,c Shengyan Liu,d Ying Sheng,a,b Karl-Gustav Rueggeberg,c Hui Wang,a Jie Li,b Frank X. Gu,d Zengtao Zhong,a Biao Kan,b,e Jun Zhua,c

The viscoelastic mucus layer of gastrointestinal tracts is a host defense barrier that a successful enteric pathogen, such as Vibrio cholerae, must circumvent. V. cholerae, the causative agent of cholera, is able to penetrate the mucosa and colonize the epithelial surface of the small intestine. In this study, we found that mucin, the major component of mucus, promoted V. cholerae movement on semisolid medium and in liquid medium. A genome-wide screen revealed that Vibrio polysaccharide (VPS) production was inversely correlated with mucin-enhanced motility. Mucin adhesion assays indicated that VPS bound to mucin. Moreover, we found that vps expression was reduced upon exposure to mucin. In an infant mouse colonization model, mutants that overexpressed VPS colonized less effectively than wild-type strains in more distal intestinal regions. These results suggest that V. cholerae is able to sense mucosal signals and modulate vps expression accordingly so as to promote fast motion in mucus, thus allowing for rapid spread throughout the intestines.

V

ibrio cholerae, a highly motile, Gram-negative curved rod bacterium, is the causative agent of cholera, an acute dehydrating diarrhea. V. cholerae has figured prominently in the history of infectious diseases as a cause of periodic global epidemics, and V. cholerae infections still occur widely in many developing countries (1, 2). Between epidemics, V. cholerae lives in natural aquatic habitats in association with various plankton and zooplankton, often in the form of biofilms. Several studies have suggested that biofilm-mediated attachment to abiotic and biotic surfaces may be important for the survival of V. cholerae in the environment (3, 4). The vps genes were previously reported to be required for exopolysaccharide biosynthesis and biofilm formation (3–5) and are positively regulated by VpsR and VpsT (6, 7). Human infection normally begins with the oral ingestion of food or water contaminated with V. cholerae. V. cholerae that survives the gastric acid shock penetrates the mucus layer and adheres to and colonizes the intestinal epithelium. As it colonizes the small intestine, V. cholerae produces an array of virulence factors, including cholera toxin (CT) and toxin-coregulated pili (TCP) (8, 9). The TCP, a type IV pilus, is considered to be the most important colonization factor because it is absolutely required for intestinal colonization. During the transition from the aquatic environment to the human body, the bacterial cells are exposed to a series of environmental changes, such as altered temperature and osmolarity, and to antimicrobial agents in the intestinal environment, such as bile and defensins (9). One of the obstacles V. cholerae has to overcome during intestinal colonization is mucus. Mucus prevents many particles, including microbes, from penetrating to the epithelial surface by sticking to these particles through multiple low-affinity hydrophobic interactions (10). The major components of mucus are mucins, which are large, highly glycosylated proteins (11). A recent study indicated that host mucins play a key role in limiting intestinal colonization by V. cholerae (12). It has been reported that V. cholerae-encoded GbpA (a chitin-binding protein), HapA

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(a Zn2⫹-dependent metalloprotease), and TagA (a mucinase) are implicated in mucin binding and penetration (13–15). However, the exact mechanisms through which V. cholerae circumvents the mucin barrier remain elusive. There exist countless examples of bacterial pathogens overcoming the mucin barrier. In Pseudomonas aeruginosa, commonly associated with nosocomial infections, mucin promotes rapid surface motility, possibly due to the lubricant properties of mucin (16). Furthermore, in Helicobacter pylori, mucin modulates cell proliferation and virulence gene expression through direct interaction with bacterial surface structures (17). Lastly, during lung infections with Staphylococcus aureus, the addition of mucin results in enhanced virulence within the host (18). It is apparent that these pathogens have adapted to recognize the mucin layer as a potential colonization site within the host and alter their gene expression accordingly. In this study, we found that mucin promotes the motility of V. cholerae. A genetic screen revealed that disruption of the Vibrio polysaccharide (VPS) synthesis pathway increased the motility of V. cholerae in mucin. We further showed that vps expression was reduced in response to

Received 27 October 2014 Returned for modification 23 November 2014 Accepted 28 December 2014 Accepted manuscript posted online 5 January 2015 Citation Liu Z, Wang Y, Liu S, Sheng Y, Rueggeberg K-G, Wang H, Li J, Gu FX, Zhong Z, Kan B, Zhu J. 2015. Vibrio cholerae represses polysaccharide synthesis to promote motility in mucosa. Infect Immun 83:1114 –1121. doi:10.1128/IAI.02841-14. Editor: A. Camilli Address correspondence to Jun Zhu, [email protected]. Z.L. and Y.W. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.02841-14. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.02841-14

Infection and Immunity

March 2015 Volume 83 Number 3

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Department of Microbiology, Nanjing Agricultural University, Nanjing, Chinaa; State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, Chinab; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USAc; Department of Chemical Engineering, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, Canadad; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Chinae

VPS Affects Motility in Mucin

mucin, and this regulation may contribute substantially to the spatial distribution of V. cholerae colonization along the intestinal tract. MATERIALS AND METHODS


RESULTS

Mucin promotes V. cholerae motility on semisolid agar plates. To investigate the effect of mucin on V. cholerae motility, we first compared the motilities of different V. cholerae strains on semisolid LB agar plates in the absence and presence of 0.4% mucin purified from pigs (Sigma). We found that most El Tor, Classic, O139, and non-O1, non-O139 strains tested displayed increased motility on plates containing mucin (Fig. 1A). The medium composition affected V. cholerae motility in mucin. The mucin effects were diminished when V. cholerae was grown in rich brain heart infusion (BHI) medium and were augmented in minimal medium (Fig. 1B). We also found that V. cholerae swam faster in mucin when glucose was used as a carbon source and that the addition of certain amino acids had some effects on V. cholerae motility in mucin (Fig. 1C and D). We also isolated mucin components from mouse small intestines and found that they had effects on V. cholerae motility similar to those of mucin purified from pigs (Fig. 1E). Taken together, these results suggest that mucin may promote motility in various V. cholerae strains under certain nutrient conditions. Genetic screens revealed that polysaccharide synthesis is involved in mucin-promoted motility. To investigate how V. cholerae promotes its motility in response to the mucin signal, we first examined the effects of mucin on the motility of V. cholerae strains with mutations in genes involved in motility, chemotaxis, or pathogenesis, or encoding predicted extracellular proteins. The El Tor C6706 strain was selected for this purpose. As predicted, flagellin and flagellar motor mutants (the flaA and motY mutants, respectively) were impaired in swimming on semisolid agar plates


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Bacterial strains and growth conditions. V. cholerae El Tor C6706 (19) was used as the wild-type strain in this study unless otherwise noted. In-frame deletions were constructed by cloning the regions flanking the target genes into suicide vector pWM91 containing a sacB counterselectable maker (20). A strain overexpressing vpsR [vpsR(Con)] was created by cloning Ptac-vpsR into a sacB-based suicide vector containing a V. cholerae intergenic fragment between VCA0104 and VCA0105 and introducing this vector into V. cholerae (21). Double-crossover recombinant mutants were selected using sucrose plates. Green fluorescent protein (GFP)-labeled V. cholerae strains were constructed by inserting Plac-gfp into the lacZ locus (22). All plasmids and oligonucleotide sequences in the present study are available upon request. Strains were propagated in LB containing appropriate antibiotics at 37°C, unless otherwise noted. Modified LB (used where indicated) contains 2‰ tryptone, 1‰ yeast extract, and 2‰ NaCl and is buffered with phosphate-buffered saline (PBS). Amino acids (Sigma) were dissolved in water and were used at a final concentration of 12.5 mM. Crude intestinal mucin was harvested as follows: segments of small intestines from adult CD1 mice were washed with PBS, and the crude mucin was scraped from the intestines into PBS. The samples were then centrifuged at 1,200 rpm for 10 min at 4°C, and the supernatants contained partially purified mucin. Examination of the effects of mucin on V. cholerae motility by using semisolid plates. The surface movement of V. cholerae was examined on modified LB containing 0.25% (wt/vol) agar and 0.4% (wt/vol) sterilized porcine mucin (type II) (Sigma) (16). For sterilization, dry powdered mucin was placed in a flask, covered with 95% ethyl alcohol, and heated at 70°C for 24 h. Sterile mucin was obtained by evaporating the alcohol. The plates were air dried for 1 h, spotted with 1 ␮l mid-logarithmic-phase cultures, and incubated at 37°C for 6 h. The surface motility zone was then measured. To test the effects of the carbon source, glucose was replaced by an equal amount of the carbon source of interest, with aspartate provided as the nitrogen source. To test the effects of the nitrogen source, total amino acids were replaced with an equal amount of a single amino acid. Genome-wide screening of genes involved in mucin-dependent motility. Wild type V. cholerae was mutagenized by a mariner transposon (Tn) carried on pNJ17 (23). Mid-log-phase cultures of mixed Tn libraries (each library contained approximate 105 independent transposon insertion mutants) were spotted at the centers of semisolid mucin plates and were incubated at 37°C for 6 h. Mutants from the edge of the motility zone were collected, and the screens were repeated two more times. The enriched mutants were then tested individually on semisolid plates without and with mucin. Mutants that displayed a hypermotile phenotype in the presence of mucin but swam normally in the absence of mucin were selected for further analysis. The transposon insertion was determined by arbitrary PCR and DNA sequencing (24). Live-cell imaging of V. cholerae motility in mucin. The wild-type, vpsA mutant, and vpsR(Con) mutant strains harboring a constitutively expressed GFP marker were grown in modified LB medium in the absence or presence of 0.4% mucin at 37°C for 12 h. The cultures were diluted 10-fold into PBS buffer and were mixed gently. The samples were then loaded into 35-mm glass-bottom dishes, and bacterial cell movements were visualized using a Nikon spinning-disk confocal microscope (⫻1,000 magnification) and an UltraVIEW VoX live-cell imaging system (PerkinElmer). The images were recorded at 1-s intervals for 1 min. Volocity 3-dimensional (3D) image analysis software (PerkinElmer) was used to analyze the data and to calculate the velocity by measuring the distance traveled by each cell in a given time. In vitro mucin adhesion assays. Mucin adhesion was calculated as the amount of mucin adsorbed per 109 bacterial cells. The wild-type, vpsA mutant, and vpsR(Con) mutant strains were grown on LB plates at 37°C

overnight. Bacterial cells were then collected and were resuspended in 1 ml PBS. The suspension was mixed with 1 ml of mucin solution (1 mg/ ml), and the mixture was incubated at 37°C for 1 h. The mixture was then centrifuged at 15,000 rpm for 1 h, and free mucin in the supernatant was quantified using the periodic acid-Schiff (PAS) staining method (25). Mucin adsorption was calculated by subtracting the free mucin concentration from the initial mucin concentration. qRT-PCR measurement of transcription of vps genes. Mid-logphase wild-type cultures were added to LB alone or to LB containing 0.04% mucin and were incubated at 37°C for 1 h. Total RNA was isolated using TRIzol (Invitrogen) and was treated with DNase I (Invitrogen) to remove contaminating genomic DNA. Three micrograms of total RNA was combined with 0.5 ␮M deoxynucleoside triphosphates (dNTPs), SUPERase-In (500 U/ml; Ambion), and 10 ␮M dithiothreitol (DTT) in the reaction buffer and was reverse transcribed with SuperScript III reverse transcriptase (Invitrogen). Reverse transcription-quantitative PCR (qRT-PCR) was carried out by using the CFX96 real-time PCR system (Bio-Rad) and a two-step RT-qPCR kit with SYBR green detection (TaKaRa). The fold change in gene transcription relative to the transcription of the housekeeping gene recA was determined using the comparative threshold cycle (CT) method. Spatial distribution of V. cholerae colonization in the infant mouse model. For in vivo competition assays using the infant mouse model (26), approximately 105 vpsR(Con) or vpsA mutant cells (lacZ⫹) were mixed with an equal number of wild-type cells (lacking lacZ), and the mixtures were inoculated intragastrically into 5-day-old infant mice. After 24 h, the animals were euthanized, and the small intestines were removed, divided into three parts of equal length (proximal, medial, and distal), and homogenized. V. cholerae cells were counted on LB plates containing 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal). This study was carried out in strict accordance with the animal protocols that were approved by the Committee on the Ethics of Animal Experiments of the Nanjing Agricultural University.

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for 6 h, unless otherwise noted. The diameter of the motility zone was measured, and the ratio of the size of the motility zone in the presence of mucin to that in the absence of mucin was calculated. (A) Motilities of different V. cholerae strains from different serogroups (see Table S2 in the supplemental material) grown in modified LB medium. Filled symbols represent strains that displayed statistical differences in motility between the presence of mucin and the absence of mucin. Horizontal lines indicate the means of motility ratios. (B) V. cholerae C6706 was grown in LB, BHI (brain heart infusion), or MM (minimal M9 medium) for 18 h. Data are means ⫾ standard deviations for three independent experiments. An asterisk indicates a statistically significant difference between results in the presence of mucin and those in the absence of mucin (P, ⬍0.05 by the Student t test). NS, no significance. (C and D) V. cholerae C6706 was grown in modified LB supplied either with 0.5% glucose, succinate, citric acid, or mannitol (C) or with 12.5 mM different amino acids (D). NRES is a 4-amino-acid combination. (E) Porcine mucin (0.4%; Sigma) or 0.4% mucin isolated from mouse small intestines was added.

both in the absence and in the presence of mucin (see Table S1 in the supplemental material). V. cholerae has three cheA genes, and cheA2 has been shown to be essential for chemotaxis (27). We found that deletion of cheA2 reduced V. cholerae motility both without and with mucin, whereas the other two cheA genes had no effect on motility. Pathogenesis-related genes and quorum-sensing regulators were not involved in mucin-modulated bacterial motility (see Table S1). In addition, we examined a number of hypothetical extracellular proteins. Although four of these mutants showed changed motility in the absence of mucin, none of them displayed altered swimming ability in the presence of mucin. Since our study of a limited number of defined mutations failed to identify a gene whose deletion affects mucin-dependent motility, we performed a genome-wide screen to look for genes that may be involved in the V. cholerae-mucin interaction. We randomly mutagenized wild-type V. cholerae by using a mariner transposon and applied mixed-transposon-library cells to the centers of swimming plates containing mucin. After a 6-h incubation, we picked mutants from the outskirts of the motility zones. After three rounds of enrichment, we obtained 6 mutants that displayed a hypermotile phenotype in the presence of mucin but swam normally in the absence of mucin. Sequence analysis revealed that 4 of these mutants had a transposon inserted into the vps locus

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(Fig. 2A), implying that VPS synthesis negatively regulates V. cholerae mobility in mucin. To confirm this, we constructed an inframe deletion in vpsA, the first gene of the vps locus. We found that the vpsA mutant displayed normal motility on nonmucin plates but that the size of its motility zone was significantly increased on mucin plates (Fig. 2B and C). This phenotype was not due to a growth rate difference between the wild type and the vpsA mutant. Figure S1 in the supplemental material shows that the wild type and the vpsA mutant multiplied similarly both in the absence and in the presence of mucin. To further demonstrate the relationship between VPS production and motility in mucin, we overexpressed the key vps activator VpsR in the wild type (6, 28). The resulting strain [vpsR(Con)] showed a reduced motility zone on mucin plates compared to those of the wild type and the vpsA mutant on mucin plates (Fig. 2B and C). Overexpression of vpsR did not affect the growth of V. cholerae; this mutant grew at a rate similar to that of the wild type both in the absence and in the presence of mucin (see Fig. S1). Taken together, these data indicate that VPS production inhibits V. cholerae motility on mucin plates. The velocity of V. cholerae in mucin is inversely correlated with VPS production. All the experiments described above were performed on semisolid plates containing 0.25% agar. To better




FIG 1 Mucin affects the motility of V. cholerae. V. cholerae strains were grown in semisolid agar (0.25%) plates in the absence or presence of 0.4% mucin at 37°C


netic structure of the Vibrio polysaccharide synthesis (vps) locus (29). Arrowheads point to the locations of the transposon insertions that affected V. cholerae motility in mucin. (B) Motilities of wild-type (wt), vpsA mutant, and vpsR-overexpressing mutant [vpsR(Con)] cells in semisolid plates in the absence (⫺) and in the presence (⫹) of 0.4% mucin. The plates were incubated at 37°C for 6 h. (C) Diameters of motility zones in the experiments for which images are shown in panel B. Data are means ⫾ standard deviations from three independent experiments. Asterisks indicate significant differences (P, ⬍0.05 by the Student t test).

mimic mucin in the intestinal environment, we examined the motility of V. cholerae cells in liquid medium with or without mucin by using a live-cell imaging system. Individual wild-type, vpsA mutant, and vpsR(Con) mutant cells containing a constitutively expressed gfp gene inserted into the lacZ locus were tracked under a microscope for 60 s, and the moving track was plotted (Fig. 3A). It is apparent that V. cholerae cells were significantly more motile in liquid medium with mucin than in the same medium without mucin. When the velocities of different cells were measured, we found that on average, vpsA mutant cells were faster than wildtype cells and vpsR(Con) mutant cells were slower (Fig. 3B). These data suggest that mucin affects the motility of V. cholerae in the liquid medium just as it does on the semisolid medium and that the velocity of V. cholerae in mucin is inversely correlated with VPS production. More mucin binds to VPS-producing cells than to non-VPSproducing cells. In order to understand why bacteria that produce more VPS display slower motility in mucin, we examined the mucin-VPS interaction by comparing the mucin adhesion abilities of wild type, vpsA mutant, and vpsR(Con) mutant cells. The vpsA mutant did not form biofilms, whereas the vpsR-overex-


DISCUSSION

During infection, V. cholerae has to penetrate the mucosal layers of the host small intestine in order to colonize the epithelial surface. In this study, we found that the motility of V. cholerae cells is increased upon exposure to mucin, the major component of mucus. Through a genetic screen, we discovered that Vibrio polysaccharide (VPS) synthesis plays a negative role in mucin-enhanced


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FIG 2 Relationship between VPS production and motility in mucin. (A) Ge-

pressing strain formed thicker biofilms than the wild type (Fig. 4A). These data are consistent with previous publications (6, 29) and indicate that these strains produce different amounts of VPS. We then incubated cells that had different amounts of VPS associated with mucin and determined how much mucin binds to cells. We found that under the test condition, approximately 0.7 mg mucin was bound to 109 wild-type cells (Fig. 4B). The mucin adhesion capacity of the vpsA mutant was significantly reduced from that of the wild type, while the mucin adhesion capacity of the vpsR(Con) mutant was increased (Fig. 4B). These data suggest that mucin may bind to polysaccharides produced by V. cholerae in a VPS concentration-dependent fashion. The interaction between mucin and VPS may produce a substantial force to slow down V. cholerae motility in mucin. Inhibition of vps expression by mucin promotes distal colonization by V. cholerae. We have shown that V. cholerae migrates faster in mucin-containing medium than in medium without mucin and that vpsA mutant cells swim faster than wild-type cells. How mucin affects vps expression is not known. We therefore performed qRT-PCR on wild-type cells to compare vps expression in the presence and in the absence of mucin. Figure 5A shows that vpsA expression was reduced ⬎2-fold when V. cholerae was exposed to 0.04% mucin for 1 h. Since vpsA is activated by VpsR and VpsT (6, 7), we also examined the expression of vpsR and vpsT in the presence of mucin. We found that both vpsR transcription and vpsT transcription were inhibited upon exposure to mucin (Fig. 5A). Based on these findings, we hypothesized that when V. cholerae enters the small intestine of the host, it may sense a certain component(s) by unknown mechanisms through VpsR and VpsT, which leads to the downregulation of vps expression and to faster migration in the mucosa, ultimately enabling the bacterium to colonize the distal small intestine efficiently (Fig. 5B). To test this hypothesis, we decided to use the infant mouse model for comparison of the colonization abilities of the wild-type, vpsA mutant, and vpsR(Con) mutant strains. We reasoned that if V. cholerae vps expression is repressed in vivo by mucosal signals, the loss of such regulation (i.e., vpsR is constitutively expressed) may have adverse effects on colonization. Figure 5C shows that for infant mice coinoculated with wild-type and vpsR(Con) cells, the CFU counts of the two strains recovered from proximal regions of the small intestine at 24 h postinfection were similar; however, fewer vpsR(Con) cells than wild-type cells colonized the medial and distal regions of the small intestine. On the other hand, the vpsA mutant displayed no colonization defects. These data suggest that regulation of polysaccharide synthesis in response to mucosal signals may contribute to the spatial distribution of V. cholerae colonization along the intestinal tract. Of note, a previous report using a rugose variant of V. cholerae has shown that vps is important for the colonization of infant mice (29). It is possible that the different strain background resulted in this discrepancy. The vps genes of C6706 have been shown previously not to be required for the colonization of infant mice (30).

Downloaded from http://iai.asm.org/ on March 23, 2015 by SUNY HEALTH SCIENCES CENTER FIG 3 Live-cell imaging of V. cholerae motility in mucin. Wild-type (wt), vpsA mutant, and vpsR(Con) mutant cells harboring a constitutively expressed GFP marker were grown in modified LB medium in the absence or in the presence of 0.4% mucin. The cells were loaded onto 35-mm glass-bottom dishes, and their movements were visualized using a Nikon spinning-disk confocal microscope and an UltraVIEW VoX system (PerkinElmer). (A) The images were recorded at 1-s intervals for 1 min. (B) Volocity 3D image analysis software (PerkinElmer) was used to analyze the data and to calculate the velocity by measuring the distance traveled by each cell in a given time. Each red horizontal bar indicates the mean of the velocities. NS, no significance. Asterisks indicate significant differences (P, ⬍0.05 by the Student t test).

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type, vpsA mutant, and vpsR(Con) mutant strains. The strains were grown in LB medium at room temperature for 24 h. Biofilm formation was visualized by staining with 0.3% crystal violet (30). (B) The suspension of wild-type, vpsA mutant, or vpsR(Con) mutant cells was mixed with 1 ml of mucin solution (1 mg/ml) and was incubated at 37°C for 1 h. The free mucin in the supernatant was quantified using the PAS staining method (25). Mucin adsorption was calculated by subtracting the free mucin concentration from the initial mucin concentration. Data are means ⫾ standard deviations for three independent experiments. Asterisks indicate significant differences (P, ⬍0.05 by the Student t test).

FIG 5 VPS production affects V. cholerae colonization. (A) vps expression in

motility, possibly due to the physical entanglement of VPS and mucin through hydrogen bonds and van der Waals interactions (31). We also found that the level of vps expression was reduced in the presence of mucin. Constitutive expression of vps in V. cholerae led to reductions in the levels of bacterial colonization of distal segments of the small intestine in the infant mouse model. These results suggest that V. cholerae is capable of modulating its gene expression profile in response to mucosal signals so as to migrate efficiently through mucus and find suitable niches to colonize in hosts. This underscores the importance of fine-tuned gene expression in enabling V. cholerae to adapt to altered environmental conditions quickly. VPS is the major component of V. cholerae biofilms. It has been postulated that V. cholerae may enter hosts while in a biofilm (30, 32) and that the bacteria need to disperse from biofilms in order to colonize intestines efficiently (30, 33). Recently, we showed that the bile salt taurocholate promotes the dispersal of V. cholerae biofilms, possibly by degrading the VPS matrix (34). The level of vps expression in the dispersed cells (planktonic state) is likely lower than that in biofilms, and vps expression is further repressed upon the sensing of mucin components, which results in fast motility in mucus. Exactly how V. cholerae penetrates mucin is not known. We show that flagellar mutants are nonmotile on plates containing mucin (see Table S1 in the supplemental material). A previous report using a mucin column showed that V. cholerae becomes nonflagellated during mucin penetration and that flagella are required only for the initial entrance into mucus (24). We suspect that the mechanism of motility on mucin-containing plates may be different from that in mucin columns. Further studies are required to understand how V. cholerae moves in mucus.


the presence of mucin. Wild-type V. cholerae was incubated in the absence and in the presence of 0.04% mucin for 1 h. RNA was isolated and was subjected to qRT-PCR analysis of vpsA, vpsR, and vpsT transcription. The changes in the transcription of these genes were calculated by normalizing the number of transcripts in the presence of mucin to that in the absence of mucin. Data are means ⫾ standard deviations for three independent experiments. Asterisks indicate statistically significant differences between transcription in the presence (⫹) of mucin and transcription in the absence (⫺) of mucin (P, ⬍0.05 by the Student t test). (B) Working model of vps expression affecting V. cholerae motility in the intestinal tract. (C) Colonization of the small intestine by wildtype and vpsA mutant strains (left) and by wild-type and vpsR(Con) mutant strains (right) in infant mice. After a 24-h incubation, the small intestines were divided into three approximately equal lengths, which were homogenized separately in order to quantify the number of V. cholerae cells colonizing each segment. The competitive index is calculated as the output ratio of the mutant to the wild type normalized to the input ratio of the mutant to the wild type. Asterisks indicate significant differences (P, ⬍0.05). NS, no significance.

The host produces mucus that covers the gastrointestinal tract to protect mucosal epithelial cells from exposure to potential enteric pathogens. However, enteric pathogens have evolved to recognize mucosal environmental cues and have developed a range of strategies to subvert the mucus barrier (35). We show here that V. cholerae reduces vps expression in order to promote fast movement in mucin. Many pathogens produce mucin-degrading enzymes to avoid aggregation by mucus (see, e.g., references 36 and 37). Helicobacter pylori can alter the pH of mucus to decrease the viscoelasticity of mucin (38). Some pathogens avoid the mucus barrier by entering the intestinal mucosa via the M cells (39). Furthermore, mucus may serve as a chemoattractant for particular enteric pathogens, which use it as an environmental cue to regulate pathogenesis-related genes (40). Interestingly, through a


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FIG 4 VPS-mucin interaction in vitro. (A) Biofilm formation by the wild-

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ACKNOWLEDGMENTS We thank Zhan Cheng and Sun Yue for technical assistance and Mark Goulian for helpful discussions. This study is supported by an NIH/NIAID R01 grant (AI080654) (to J.Z.), a NIH EID training grant (T32AI055400) (to K.-G.R.), an NSFC general fund grant (81371763), a fellowship from the China Scholarship Council (to H.W.), the Priority Project of State Key Laboratory for Infectious Disease Prevention and Control (2014SKLID101) (to B.K.), and a Natural Sciences and Engineering Research Council (NSERC) doctoral fellowship (to S.L.).

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Motility as a virulence factor for Vibrio cholerae.

Rapid effects of a protective O-polysaccharide-specific monoclonal IgA on Vibrio cholerae agglutination, motility, and surface morphology.

Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production.

Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment.

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2.

Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models.

A cis-regulatory antisense RNA represses translation in Vibrio cholerae through extensive complementarity and proximity to the target locus.

Chromosome segregation in Vibrio cholerae.

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Structural studies of the Vibrio cholerae O:3 O-antigen polysaccharide.

Structural characterization of the extracellular polysaccharide from Vibrio cholerae O1 El-Tor.

Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome.

Letter: Isolation of Vibrio cholerae.

Siderophore production by Vibrio cholerae.

ATP-association to intrabacterial nanotransportation system in Vibrio cholerae.

Regulatory cascade controls virulence in Vibrio cholerae.

Induction of melanin biosynthesis in Vibrio cholerae.

Transposon-facilitated recombination in Vibrio cholerae.

Dynamics in genome evolution of Vibrio cholerae.

Vibrio cholerae serogroup O1 in northeast Thailand.

Vibrio cholerae persisted in microcosm for 700 days inhibits motility but promotes biofilm formation in nutrient-poor lake water microcosms.

Urinary tract infection due to NonO1 Vibrio cholerae.

Vibrio cholerae O1 Imported from Iraq to Kuwait, 2015.

Non-O1 Vibrio cholerae NRT36S produces a polysaccharide capsule that determines colony morphology, serum resistance, and virulence in mice.