Bile salts and alkaline pH reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae ToxR and ToxS.

ToxR is a transmembrane transcription factor that is essential for virulence gene expression and human colonization by Vibrio cholerae. ToxR requires its operon partner ToxS, a periplasmic integral membrane protein, for full activity. These two proteins are thought to interact through their respective periplasmic domains, ToxRp and ToxSp. In addition, ToxR is thought to be responsive to various environmental cues, such as bile salts and alkaline pH, but how these factors influence ToxR is not yet understood. Using NMR and reciprocal pull down assays, we present the first direct evidence that ToxR and ToxS physically interact. Furthermore, using NMR and DSF, it was shown that the bile salts cholate and chenodeoxycholate interact with purified ToxRp and destabilize it. Surprisingly, bile salt destabilization of ToxRp enhanced the interaction between ToxRp and ToxSp. In contrast, alkaline pH, which is one of the factors that leads to ToxR proteolysis, decreased the interaction between ToxRp and ToxSp. Taken together, these data suggest a model whereby bile salts or other detergents destabilize ToxR, increasing its interaction with ToxS to promote full ToxR activity. Subsequently, as V. cholerae alkalinizes its environment in late stationary phase, the interaction between the two proteins decreases, allowing ToxR proteolysis to proceed.

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

Proteolysis of virulence regulator ToxR is associated with entry of Vibrio cholerae into a dormant state.

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state.

The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms.

FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms.IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor that coordinately controls the pathways of FA degradation and biosynthesis in enteric bacteria. This study identifies a new link between FA metabolism and virulence in the El Tor biotype by showing that FadR influences both the transcription and posttranslational regulation of the master virulence regulator ToxT by two distinct mechanisms.

Identification of a Small Molecule Activator for AphB, a LysR-Type Virulence Transcriptional Regulator in Vibrio cholerae.

AphB is a LysR-type transcriptional regulator (LTTR) that cooperates with a second transcriptional activator, AphA, at the tcpPH promoter to initiate expression of the virulence cascade in Vibrio cholerae. Because it is not yet known whether AphB responds to a natural ligand in V. cholerae that influences its ability to activate transcription, we used a computational approach to identify small molecules that influence its activity. In silico docking was used to identify potential ligands for AphB, and saturation transfer difference nuclear magnetic resonance was subsequently employed to access the validity of promising targets. We identified a small molecule, BP-15, that specifically binds the C-terminal regulatory domain of AphB and increases its activity. Interestingly, molecular docking predicts that BP-15 does not bind in the putative primary effector-binding pocket located at the interface of RD-I and RD-II as in other LTTRs, but rather at the dimerization interface. The information gained in this study helps us to further understand the mechanism by which transcriptional activation by AphB is regulated by suggesting that AphB has a secondary ligand binding site, as observed in other LTTRs. This study also lays the groundwork for the future design of inhibitory molecules to block the V. cholerae virulence cascade, thereby preventing the devastating symptoms of cholera infection.

Intestinal Colonization Dynamics of Vibrio cholerae.

To cause the diarrheal disease cholera, Vibrio cholerae must effectively colonize the small intestine. In order to do so, the bacterium needs to successfully travel through the stomach and withstand the presence of agents such as bile and antimicrobial peptides in the intestinal lumen and mucus. The bacterial cells penetrate the viscous mucus layer covering the epithelium and attach and proliferate on its surface. In this review, we discuss recent developments and known aspects of the early stages of V. cholerae intestinal colonization and highlight areas that remain to be fully understood. We propose mechanisms and postulate a model that covers some of the steps that are required in order for the bacterium to efficiently colonize the human host. A deeper understanding of the colonization dynamics of V. cholerae and other intestinal pathogens will provide us with a variety of novel targets and strategies to avoid the diseases caused by these organisms.

Effects of tcpB Mutations on Biogenesis and Function of the Toxin-Coregulated Pilus, the Type IVb Pilus of Vibrio cholerae.

UNLABELLED: Vibrio cholerae is the etiological agent of the acute intestinal disorder cholera. The toxin-coregulated pilus (TCP), a type IVb pilus, is an essential virulence factor of V. cholerae Recent work has shown that TcpB is a large minor pilin encoded within the tcp operon. TcpB contributes to efficient pilus formation and is essential for all TCP functions. Here, we have initiated a detailed targeted mutagenesis approach to further characterize this salient TCP component. We have identified (thus far) 20 residues of TcpB which affect either the steady-state level of TcpB or alter one or more TCP functions. This study provides a solid framework for further understanding of the complex role of TcpB and will be of use upon determination of the crystal structure of TcpB or related minor pilin orthologs of type IVb pilus systems. IMPORTANCE: Type IV pili, such as the toxin-coregulated pilus (TCP) in V. cholerae, are bacterial appendages that often act as essential virulence factors. Minor pilins, like TcpB, of these pili systems often play integral roles in pilus assembly and function. In this study, we have generated mutations in tcpB to determine residues of importance for TCP stability and function. Combined with a predicted tertiary structure, characterization of these mutants allows us to better understand critical residues in TcpB and the role they may play in the mechanisms underlying minor pilin functions.

Role of ToxS in the proteolytic cascade of virulence regulator ToxR in Vibrio cholerae.

Two of the primary virulence regulators of Vibrio cholerae, ToxR and TcpP, function together with cognate effector proteins. ToxR undergoes regulated intramembrane proteolysis (RIP) during late stationary phase in response to nutrient limitation at alkaline pH; however, the specific function of its cognate ToxS remains unresolved. In this work, we found that ToxR rapidly becomes undetectable in a ΔtoxS mutant when cultures are exposed to either starvation conditions or after alkaline pH shock individually. A ΔtoxS mutant enters into a dormant state associated with the proteolysis of ToxR at a faster rate than wild-type, closely resembling a ΔtoxR mutant. Using a mutant with a periplasmic substitution in ToxS, we found that the proteases DegS and DegP function additively with VesC and a novel protease, TapA, to degrade ToxR in the mutant. Overall, the results shown here reveal a role for ToxS in the stabilization of ToxR by protecting the virulence regulator from premature proteolysis.

Secretion of TcpF by the Vibrio cholerae toxin-coregulated pilus biogenesis apparatus requires an N-terminal determinant.

Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81-92, 2003). This phenomenon is not unique to V. cholerae; secreted virulence factors that are dependent on the presence of components of the type IV pilus biogenesis apparatus for secretion have been reported with Dichelobacter nodosus (R. M. Kennan, O. P. Dhungyel, R. J. Whittington, J. R. Egerton, and J. I. Rood, J. Bacteriol. 183:4451-4458, 2001) and Francisella tularensis (A. J. Hager et al., Mol. Microbiol. 62:227-237, 2006). Using site-directed mutagenesis, we demonstrated that the secretion of TcpF is dependent on the presence of selected amino acid R groups at position five. We were unable to find other secretion determinants, suggesting that Y5 is the major secretion determinant within TcpF. We also report that proteins secreted in a type IV pilus biogenesis apparatus-dependent manner have a YXS motif within the first 15 amino acids following the Sec cleavage site. The YXS motif is not present in proteins secreted by type II secretion systems, indicating that this is unique to type IV pilus-mediated secretion. Moreover, we show that TcpF interacts with the pilin TcpA, suggesting that these proteins are secreted by the type IV pilus biogenesis system. These data provide a starting point for understanding how type IV pili can mediate secretion of virulence factors important for bacterial pathogenesis.

Characterization of BreR interaction with the bile response promoters breAB and breR in Vibrio cholerae.

The Vibrio cholerae BreR protein is a transcriptional repressor of the breAB efflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol. 190:7441-7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at the breAB promoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in the breAB promoter. However, only one of these sites, the proximal 29-bp site, is necessary for BreR-mediated transcriptional repression of breAB expression. In addition, it was determined that BreR represses its own expression by recognizing a 28-bp site at the breR promoter. These sites comprise regions of dyad symmetry within which residues critical for BreR function could be identified. The BreR consensus sequence AANGTANAC-N(6)-GTNTACNTT overlaps the -35 region at both promoters, implying that the repression of gene expression is achieved by interfering with RNA polymerase binding at these promoters.

The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH.

Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression. We present here the 2.2 Å X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA. We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.

The major Vibrio cholerae autoinducer and its role in virulence factor production.

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signalling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. Here we describe the purification of CAI-1 and identify the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We provide a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluate their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus). These findings suggest that CAI-1 could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena.

Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes.

Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraC-family regulator, ToxT. We have solved the 1.9 A resolution crystal structure of ToxT, which reveals folds in the N- and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.

Protection and attachment of Vibrio cholerae mediated by the toxin-coregulated pilus in the infant mouse model.

Colonization of the human small intestine by Vibrio cholerae is an essential step in pathogenesis that requires the type IV toxin-coregulated pilus (TCP). To date, three functions of TCP have been characterized: it serves as the CTXΦ receptor, secretes the colonization factor TcpF, and functions in microcolony formation by mediating bacterium-bacterium interactions. Although type IV pili in other pathogenic bacteria have been characterized as playing a major role in attachment to epithelial cells, there are very few studies to suggest that TCP acts as an attachment factor. Taking this into consideration, we investigated the function of TCP in attachment to Caco-2 cells and found that mutants lacking TCP were defective in attachment compared to the wild type. Overexpression of ToxT, the activator of TCP, significantly increased attachment of wild-type V. cholerae to Caco-2 cells. Using field-emission scanning electron microscopy (FESEM), we also observed TCP-mediated attachment to the small intestines of infected infant mice by using antibodies specific to TCP and V. cholerae. Remarkably, we also visualized matrices comprised of TCP appearing to engulf V. cholerae during infection, and we demonstrated that these matrices protected the bacteria from a component of bile, disclosing a possible new role of this pilus in protection of the bacterial cells from antimicrobial agents. This study provides new insights into TCP's function in V. cholerae colonization of the small intestine, describing additional roles in mediating attachment and protection of V. cholerae bacterial cells.

H-NS binding and repression of the ctx promoter in Vibrio cholerae.

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at ctx. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the -35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the ctx promoter region. In addition, regulatory differences between the ctx and tcpA promoters were examined. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro. In contrast to ToxT-dependent activation of the tcpA promoter, ToxT activation of ctx did not require the C-terminal domain of the α-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of ctx and tcpA by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.

The physical basis of type 4 pilus-mediated microcolony formation by Vibrio cholerae O1.

The Vibrio cholerae toxin co-regulated pilus (TCP) is a type 4b pilus that mediates bacterial microcolony formation, which is essential for intestinal colonization. Structural analyses have defined a surface domain of the TcpA pilin subunit that is displayed repeatedly around the pilus filament surface and forms the molecular basis for pilus-pilus interactions required for microcolony formation. The physical attributes of this domain that lead to pilus-pilus association between bacteria are not known. Mutational analysis has revealed alterations within this domain that allow pilus-pilus interactions among pili expressed by individual bacteria, but do not allow pilus-pilus mediated association between bacteria. We characterized these altered strains using conventional microscopy, as well as three-dimensional high-resolution field emission scanning electron microscopy (FESEM), to reveal the physical difference between nonproductive and productive pilus associations that lead to interactions among multiple bacteria and result in microcolony formation. These findings pave the way towards investigation of the biophysical parameters involved in this basic bacterial property that promotes colonization of intestinal and other biological surfaces.

Vibrio cholerae El Tor TcpA crystal structure and mechanism for pilus-mediated microcolony formation.

Type IV pili (T4P) are critical to virulence for Vibrio cholerae and other bacterial pathogens. Among their diverse functions, T4P mediate microcolony formation, which protects the bacteria from host defences and concentrates secreted toxins. The T4P of the two V. cholerae O1 disease biotypes, classical and El Tor, share 81% identity in their TcpA subunits, yet these filaments differ in their interaction patterns as assessed by electron microscopy. To understand the molecular basis for pilus-mediated microcolony formation, we solved a 1.5 A resolution crystal structure of N-terminally truncated El Tor TcpA and compared it with that of classical TcpA. Residues that differ between the two pilins are located on surface-exposed regions of the TcpA subunits. By iteratively changing these non-conserved amino acids in classical TcpA to their respective residues in El Tor TcpA, we identified residues that profoundly affect pilus:pilus interaction patterns and bacterial aggregation. These residues lie on either the protruding d-region of the TcpA subunit or in a cavity between pilin subunits in the pilus filament. Our results support a model whereby pili interact via intercalation of surface protrusions on one filament into depressions between subunits on adjacent filaments as a means to hold V. cholerae cells together in microcolonies.

Nutrient-dependent, rapid transition of Vibrio cholerae to coccoid morphology and expression of the toxin co-regulated pilus in this form.

The acute diarrhoeal disease cholera is caused by the aquatic pathogen Vibrio cholerae upon ingestion of contaminated food or water by the human host. The mechanisms by which V. cholerae is able to persist and survive in the host and aquatic environments have been studied for years; however, little is known about the factors involved in the adaptation or response of V. cholerae transitioning between these two environments. The transition from bacillary to coccoid morphology is thought to be one mechanism of survival that V. cholerae uses in response to environmental stress. Coccoid morphology has been observed for V. cholerae while in a viable but non-culturable (VBNC) state, during times of nutrient limitation, and in the water-diluted stool of cholera-infected patients. In this study we sought conditions to study the coccoid morphology of V. cholerae, and found that coccoid-shaped cells can express and produce the virulence factor toxin co-regulated pilus (TCP) and are able to colonize the infant mouse to the same extent as bacillus-shaped cells. This study suggests that TCP may be one factor that V. cholerae utilizes for adaptation and survival during the transition between the host and the aquatic environment.

Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes.

Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.

A colonization factor links Vibrio cholerae environmental survival and human infection.

Many bacteria that cause diseases must be able to survive inside and outside the host. Attachment to and colonization of abiotic or biotic surfaces is a common mechanism by which various microorganisms enhance their ability to survive in diverse environments. Vibrio cholerae is a Gram-negative aquatic bacillus that is often found in the environment attached to the chitinous exoskeletons of zooplankton. It has been suggested that attachment to zooplankton enhances environmental survival of Vibrio spp., probably by providing both an abundant source of carbon and nitrogen and protection from numerous environmental challenges. On ingestion by humans, some serogroups of V. cholerae cause the diarrhoeal disease cholera. The pathophysiology of cholera is a result of the effects of cholera toxin on intestinal epithelial cells. For sufficient quantities of cholera toxin to reach the intestinal epithelium and to produce clinical symptoms, colonization of the small bowel must occur. Because most V. cholerae do not colonize humans, but all probably require strategies for survival in the environment, we considered that colonization factors selected for in the environment may be the same as those required for intestinal colonization of humans. In support of this hypothesis, here we have identified a single protein required for efficient intestinal colonization that mediates attachment to both zooplankton and human epithelial cells by binding to a sugar present on both surfaces.