Ins and Outs of cholerae Vibrio cholerae transitions between the human gut and the aquatic environment are aided by specific shifts in gene expression

Stefan Schild, Anne L. Bishop, and Andrew Camilli

he gram-negative, motile, curved- Organization reported a total of 236,896 chol- rod bacterium Vibrio cholerae can era cases with 6,311 deaths worldwide. These —an acute, explosive figures likely greatly underestimate the toll of T diarrheal disease. Epidemic cholera cholera. Many cases go unreported during sea- was described in the 1500s, but re- sonal outbreaks, and a substantial percentage of ports of cholera-like symptoms date back much people die without diagnosis. earlier, to the times of Hippocrates and the Buddha. In 1849 , a London physi- Two Serogroups Give Rise cian, determined that cholera is transmitted by to Cholera Disease water, and in 1883 successfully isolated the cholera vibrio. Facilitated by the Despite there being more than 200 known sero- expansion of global trade routes, cholera spread groups of V. cholerae in the aquatic environ- worldwide in 1817 and remains a global health ment, only strains of two serogroups, O1 and challenge. O139, are known to cause epidemic disease. Cholera patients typically lose large volumes O-antigen and core oligosaccharide of the O1 of fluid. If left untreated, even previously healthy and O139 lipopolysaccharides, as well as the adult patients may become severely dehydrated capsule of O139, contribute in colonization of and can die. Although rehydration therapy the small intestine and adhesion to epithelial makes cholera survivable, it does not cure the cells. Strains within serogroup O1 can be further or prevent . Cholera remains divided into two biotypes, and classical. a social and economic burden, especially in the These two biotypes are based upon phenotypic developing world. In 2006 the World Health traits, including phage and polymyxin B sensi- tivity or resistance. Classical and El Tor exhibit many differences, often at the level of gene regulation. Summary The genome sequence of the V. cholerae • The facultative Vibrio cholerae has clinical isolate N16961 is of similar size to Stefan Schild and adapted to survive and colonize two very differ- that of other ␥-. In this case, Anne L. Bishop are ent environments—the aquatic environment N16961 contains 4.033 Mbp, which is sim- research associates and the human gastrointestinal tract. ilar in size to that of K12 and Andrew Camilli • The intracellular signal molecule c-di-GMP can with 4.639 Mbp. However, the V. cholerae is a professor in the inversely regulate genes specific for the aquatic genome is split into two , with Howard Hughes environment or the host. one containing 2.961 Mbp and the other Medical Institute • Quorum sensing is one of the signals that helps 1.072 Mbp, a feature that is seen in other and the Depart- to regulate c-di-GMP levels. Vibrio species. Having two smaller chromo- ment of Molecular • While genes essential for colonization are in- somes, which replicate in a coordinated but Biology and Micro- duced early during , late in vivo in- independent fashion, might speed replica- biology, Tufts Uni- duced genes can increase fitness for transition tion. versity School of into the aquatic environment. V. cholerae is closely related to other Medicine, Boston, aquatic , some of which live closely Mass.

Volume 3, Number 3, 2008 / Microbe Y 131 with marine organisms either as symbionts, films. In static cultures biofilm formation is commensals, or . V. cholerae O1 dependent on biosynthesis of Vibrio exopo- likely arose from an aquatic ancestor that was lysaccharide (VPS). In contrast, in a flow cell nonpathogenic to humans. V. cholerae is not system a vps-independent biofilm has recently only transmitted via contaminated water, like been described by Alfred Spormann and col- other fecal-orally transmitted pathogens such as leagues at Stanford University, Calif. It is likely or E. coli, but also spends consider- that survival in aquatic environments is aided by able time as an aquatic organism in both fresh the formation of such multicellular structures. and water. When a human ingests V. cholerae, the bacte- Since epidemiological record-keeping began ria face a sudden shift in temperature and osmo- in the early 19th century, eight cholera pandem- larity as well as the extreme acidity of the stom- ics have been recorded. O1 classical caused the ach. Later, in the human gut, the fifth and sixth and possibly the four encounter alkaline pH, digestive enzymes, bile, earlier pandemics. In contrast, O1 El Tor is and components of the innate immune system. responsible for the seventh , which In the small intestine, the polar flagellum propels began in the 1960s. Although strains of the individual V. cholerae cells into the mucus layer classical biotype are probably extinct in nature, where they can attach to host epithelial cells, the both serotypes are still extensively studied in the major sites of bacterial proliferation. laboratory, and differences that may explain the In the mouse as well as in humans, V. cholerae emergence of El Tor remain of great interest to induces several virulence factors, including chol- researchers in the field. era toxin (CTX) and the toxin-coregulated Since its recent emergence in 1992, cholera (TCP), the major colonization factor required infections caused by O139 have spread world- for microcolony formation. In mice this gene wide. Although O139 has not replaced O1 El induction is rapid, occurring within the first five Tor, it is responsible for an eighth cholera pan- hours. The activity of CTX causes a massive demic that is running in parallel with the sev- secretory diarrhea that, if untreated, can result enth. Genetic analysis reveals O139 to be closely in hypotensive shock, organ failure, and death related to O1 El Tor, suggesting that O139 within a day. The ability of V. cholerae to repli- evolved from an El Tor O1 strain that acquired cate with a generation time of 20 minutes may new LPS and capsule biosynthesis genes. be advantageous during the gastrointestinal (GI) tract colonization stage of its life cycle, when the potent effects of CTX provide only a short time Life Cycle of Vibrio cholerae in which to proliferate. To induce these viru- In our laboratory we are striving to gain a better lence factors, V. cholerae relies on signal trans- understanding of the full life cycle (all of the “ins duction and transcriptional regulatory cascades and outs”) of the facultative pathogen V. chol- that include the two transmembrane regulators erae. As a facultative pathogen, V. cholerae re- ToxR and TcpP, as well as the cytoplasmic sides in two worlds (Fig. 1): as a natural inhab- regulator ToxT. itant of aquatic ecosystems and as the causative Cholera researchers are making great ad- agent of a severe diarrhea in the gastrointestinal vances in understanding changes in gene expres- tract of its human host. In the environment, V. sion at different stages in the V. cholerae life cholerae is found in association with zooplank- cycle. Our lab’s research has focused on identi- ton, crustaceans, and egg masses of chirono- fying in vivo-induced genes at early stages of mids, commonly called “midges.” A variety of infection, using recombination-based in vivo ex- factors that promote adherence to sur- pression technology (RIVET) in combination faces of such organisms have been reported, with the infant mouse model. Briefly, the including N-acetylglucosamine binding method relies on a library of strains carrying A (GbpA) as well as mannose-sensitive hemag- transcriptional fusions to tnpR (encoding a re- glutinin (MSHA) and PilA pili. This association solvase) that are not expressed in vitro. When a may provide an environmental advantage, since subset of these strains expresses the resolvase in V. cholerae is capable of degrading chitin and vivo, they excise a cassette harboring selectable using it as a carbon and nitrogen source. Fur- and counter-selectable markers that are used to thermore, V. cholerae O1 and O139 form bio- identify the strains. Using microarray technol-

132 Y Microbe / Volume 3, Number 3, 2008 FIGURE 1

Model for the life cycle of V. cholerae. When humans ingest or water containing V. cholerae, several bacterial genes are induced, including those encoding and toxin coregulated pilus (bottom right), while c-di-GMP levels drop (right). A scanning electron micrograph (bottom left) shows V. cholerae associated with a microvillus in the infant mouse small intestine (magnification, ϫ750; taken by Michael J. Angelichio). Inducing late genes (bottom left) may help to maintain an infection and increase fitness prior to the transition back into aquatic environments. Genes induced late during infection include DGCs, which are predicted to increase c-di-GMP level prior to release into the aquatic environment (left). After release into the environment hyperinfectious V. cholerae can infect a new human host (short-term persistence). Alternatively, V. cholerae may form associations with and biofilms upon chitinous material, facilitating long-term persistence in the aquatic environment. Pictures of the pond in Bangladesh and of a (top) were taken by Lori Bourassa.

Volume 3, Number 3, 2008 / Microbe Y 133 ogy, we and other research groups have ana- infant mouse model and can be observed pheno- lyzed the transcriptome of V. cholerae in stool typically using chemotaxis assays. The nature of from cholera patients to better understand what V. cholerae in stool samples and its effects on happens to the bacteria during human infec- cholera transmission are just beginning to be tions. A green fluorescent protein-based screen investigated. For instance rice-water stool some- allowed Jun Zhu and co-workers at University times contains V. cholerae aggregates, some of of Pennsylvania in Philadelphia to identify genes which are associated with mucus, according to downregulated in vivo. They demonstrated that Stephen B. Calderwood and colleagues at Mas- the repression of the MSHA-operon is necessary sachusetts General Hospital and Harvard Med- to evade the host immune system. Gary K. ical School in Boston, Mass., and Shah M. Fa- Schoolnik and coworkers at Stanford Univer- ruque and colleagues at the International Centre sity, Stanford, Calif., reported that once V. chol- for Diarrhoeal Disease Research in Bangladesh. erae colonize the rabbit ileal loop, they produce Such aggregates could enhance environmental a stationary-phase sigma factor (RpoS) that en- survival and affect rates of transmission. ables detachment from the epithelial surface. Recently we identified V. cholerae genes spe- Whether this sigma factor is required for detach- cifically induced late in infection using a modi- ment during infection in an open intestinal tract fied RIVET screen and the infant mouse model. is unclear. However, at some stage during the Mutant analysis revealed that most of the late infection, bacteria detach from the epithelial genes are necessary neither for survival in the layer and are shed in “rice-water” stool into the host nor for maintenance of the infection. By environment. establishing a novel transition assay, we could demonstrate that some of those late gene mu- tants, after in vivo passage, exhibit a fitness The “Ready” State of V. cholerae defect if incubated in pond water and/or rice- V. cholerae shed by cholera patients are more water stool. In vivo passage is essential to ob- infectious than laboratory-cultured bacteria serve these phenotypes. When human volun- when tested in mice. Mathematical modeling of teers ingested a RIVET library made in an human epidemiological data supports the exis- attenuated vaccine-candidate strain of V. chol- tence of a hyperinfectivity phenomenon, as re- erae, we and James Kaper and colleagues at the ported by David M. Hartley at the University University of Maryland in Baltimore identified a of Maryland School of Medicine in Baltimore similar set of late-induced genes, indicating that and his collaborators. Hyperinfectivity is main- this type of gene regulation occurs when this tained for at least5hinpond water, suggesting pathogen passes through human hosts. that expression of a subset of V. cholerae genes The transition from host to environment is as responsible for hyperinfectivity persists tempo- harsh as from the environment to the host. In rarily and could facilitate reinfection. leaving the mammalian GI tract into fresh wa- Siouxsie Wiles, Gordon Dougan, and Gad ter, V. cholerae faces a 50-fold drop in osmolar- Frankel at Imperial College London in England ity, a shift to lower temperature, and a massive have observed similar hyperinfectivity of natu- shift in environmental and nutrients. Ex- rally transmitted compared with laboratory- tensive changes in the transcriptome and pro- grown bacteria for Citrobacter rodentium after teome to adapt to the new conditions are ener- it passes through the mouse GI tract. Whether getically costly, but V. cholerae has the the mechanisms of hyperinfectivity for V. chol- advantage that, through evolution, it can “pre- erae and C. rodentium differ or are conserved pare” for this transition. That is to say, a facul- remains to be determined. However, Frankel tative pathogen like V. cholerae may induce and his collaborators also report that well-char- genes that are beneficial (and repress genes that acterized virulence genes are strongly expressed are detrimental) for environmental survival after shedding of C. rodentium by mice, whereas while still in the host, due to a selective advan- in human stool the ToxR and ToxT regulons of tage once in the aquatic environment. This idea V. cholerae are not. is supported by the identification of operons Meanwhile, our analysis indicates that repres- involved in chitin utilization and the biosynthe- sion of chemotaxis in fresh stool-borne V. chol- sis of MSHA as being induced late in infections. erae enhances virulence in a new host using the A significant percentage of the late in vivo

134 Y Microbe / Volume 3, Number 3, 2008 induced genes is associated with uptake or trans- di-GMP plays an important role in this transi- port systems. One explanation for this observa- tion. tion is that V. cholerae may need these systems The molecules that link changes in c-di-GMP ready to use and fully functional when it faces level with observed changes in gene transcrip- the aquatic environment. Expression of these tion have proved difficult to identify. Using uptake systems in vivo may allow V. cholerae to bioinformatic analysis, Dorit Amikam and Mi- hoard factors that will be limited later in the chael Galperin at the Tel-Hai Academic College environment, such as carbon sources. in Jerusalem, Israel, and National Institutes of Health in Bethesda, Md., respectively, identified the PilZ domain as a putative c-di-GMP binding V. cholerae Transitions Are Mediated motif. The PilZ-domains of cellulose synthase in by c-di-GMP Signaling Gluconacetobacter xylinus and other PilZ- V. cholerae induces diguanylate cyclases (DGCs) in E. coli and Caulobacter crescentus at the late stage of infection, which would lead mediate binding to c-di-GMP and control pro- to an increase in cyclic-di-guanylate (c-di-GMP). tein activity. c-di-GMP is an intracellular bacterial second The V. cholerae genome encodes five PilZ- messenger commonly found in gram-negative domain-containing proteins, designated PlzA- and some gram-positive organisms. DGCs syn- PlzE; among them, PlzB shows only weak ho- thesize c-di-GMP from GTP, while specific mology to the PilZ-domain consensus. We find phosphodiesterases (PDEs) degrade c-di-GMP. that both PlzD and PlzC bind c-di-GMP in vitro, V. cholerae encodes about 60 proteins contain- while a plzD PilZ-domain point mutation abro- ing a DGC, a PDE, or both domains, only a gates binding. A double plzCD mutant is re- fraction of which have been demonstrated to be duced in virulence in the murine model and this can be complemented by plzD on a , but active. not by the plzD PilZ-domain point mutant. We In V. cholerae high levels of c-di-GMP en- have yet to assign roles for the other predicted hance biofilm formation by increasing expres- Plz-proteins in the life cycle of V. cholerae, and sion of vps genes, while inhibiting we do not understand the molecular mechanism of virulence and motility genes. In the classical by which Plz proteins transduce c-di-GMP sig- biotype, mutation of vieA, which is a response nals. Further, because DGCs are found in organ- regulator and PDE, elevates c-di-GMP resulting isms that lack any predicted PilZ-domain pro- in enhanced biofilm and dramatically reduced teins, other classes of proteins likely bind and motility and virulence. Furthermore, transcrip- respond to c-di-GMP. tional changes in a vieA mutant are similar to Recently, an exciting link has been identified those seen with ectopic DGC overexpression. between c-di-GMP signaling and bacterial cell These data show that vieA is essential for c- density. Quorum sensing allows bacteria to di-GMP down-regulation in the classical bio- sense their density and/or the density of other type, yet in the El Tor biotype, vieA deletion bacteria in their vicinity. In many other patho- mutants show no detectable phenotypes and genic bacteria at high density, quorum sensing relatively minor changes in gene transcription. activates virulence gene expression (enterohae- This is one of many intriguing differences be- morrhagic E. coli) and/or biofilm formation tween O1 El Tor and classical. We suspect that (Pseudomonas). V. cholerae is surprisingly dif- one or more of the other more than 20 PDEs in ferent in that at high density a key protein in its V. cholerae are substituting for vieA in El Tor. quorum sensing cascade (HapR) inhibits both Based on such studies, we hypothesize that virulence gene expression and biofilm forma- levels of c-di-GMP in V. cholerae are high in at tion. Intriguingly, hapR is often mutated in both least some aquatic environments, facilitating environmental and clinical isolates such as the biofilm formation, but are reduced early during classical biotype strain O395. In addition, the infection to allow proper expression of the vir- binding site for HapR in the promoter of aphA, ulence genes. Our finding that a mutant strain, which is a key virulence gene regulator that is which lacks three DGCs expressed late in infec- inhibited by HapR, is mutated in classical strain tion, is less fit for transitioning from stool to O395. These observations suggest that HapR is pond water further suggests that elevated c- not essential to the V. cholerae life cycle. Low

Volume 3, Number 3, 2008 / Microbe Y 135 cell density signaling through phospho-LuxO of the “ins and outs” of V. cholerae as it passes turns off hapR, enabling virulence and/or bio- through very different environments between film genes to be expressed. Recently, Bonnie L. host and aquatic reservoirs. Bassler at Princeton University, Princeton, N.J., Research into the regulation and character- discovered a new target for quorum sensing ization of MSHA is a good example of how which is independent of hapR, the predicted information acquired by different approaches in DGC VCA0939. VCA0939 is acti- different laboratories comes together to form a vated by the same sRNAs that inhibit transla- more coherent model. Initially thought to be an tion of hapR and destabilize the hapR mRNA. adherence factor for binding to epithelial cells, Whereas hapR mutations are common in clini- MSHA was later shown to be dispensable for cal isolates, the quorum regulator RNA (qrr) colonization in vivo but important for biofilm sRNAs are highly conserved, suggesting that formation and attachment to chitinous surfaces HapR-independent, sRNA-dependent branches by Ronald K. Taylor and coworkers at Dart- of quorum sensing may be essential to V. chol- mouth Medical School, Hanover, N.H. Jun Zhu erae. This opens up exciting new fields of inves- and colleagues at the University of Pennsylvania tigation into HapR-independent quorum sens- made it evident that the expression of MSHA ing and coordination between quorum sensing has to be down-regulated in vivo to evade the and c-di-GMP signaling in the V. cholerae life host immune response. Now, we have identified cycle. MSHA biosynthesis genes as being induced in the late stage of infection, which leads to the idea that V. cholerae might, in this way, prepare Conclusions itself for the transition into the aquatic environ- In the last century improved sanitation and im- ment at the end of the infection. provements in have In summary, we hope that an improved un- been the most important breakthroughs in chol- derstanding of the V. cholerae organism at all era prevention and treatment. On the pathogen- stages of its life cycle, in both host and aquatic esis research front, the powerful combination of environments, will help us to prevent disease studies using transcriptional profiling or in vivo with novel ways to block key steps in pathogen- expression technologies, such as RIVET, is be- esis or transmission and to develop effective and ginning to create a more comprehensive picture inexpensive vaccines.

SUGGESTED READING Butler, S. M., and A. Camilli. 2005. Going against the grain: chemotaxis and infection in Vibrio cholerae. Nature Rev. Microbiol. 3:611–620. Hammer, B. K., and B. L. Bassler. 2007. Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc. Natl. Acad. Sci. USA 104:11145–1149. Hsiao, A., Z. Liu, A. Joelsson, and J. Zhu. 2006. Vibrio cholerae virulence regulator-coordinated evasion of host immunity. Proc. Natl. Acad. Sci. USA 103:14542–14547. Lombardo, M., J. Michalski, H. Martinez-Wilson, C. Morin, T. Hilton, C. G. Osorio, J. P. Nataro, C. O. Tacket, A. Camilli, and J. Kaper. 2007. An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers. Proc. Natl. Acad. Sci. USA 104:18229–18234. Nelson, E. J., A. Chowdhury, J. B. Harris, Y. A. Begum, F. Chowdhury, A. I. Khan, R. C. LaRocque, A. L. Bishop, E. T. Ryan, A. Camilli, F. Qadri, and S. B. Calderwood. 2007. Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea. Proc. Natl. Acad. Sci. USA 104:19091–19096. Nielsen, A. T., N. A. Dolganov, G. Otto, M. C. Miller, C. Y. Wu, and G. K. Schoolnik. 2006. RpoS controls the Vibrio cholerae mucosal escape response. PLoS Pathog 2:e109. Pratt, J. T., R. Tamayo, A. D. Tischler, and A. Camilli. 2007. PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae. J. Biol. Chem. 282:12860–12870. Schild, S., R. Tamayo, E. J. Nelson, F. Qadri, S. B. Calderwood, and A. Camilli. 2007. Genes induced late in infection increase fitness of Vibrio cholerae after release into the environment. Cell Host Microbe 2:264–277. Tamayo, R., J. T. Pratt, and A. Camilli. 2007. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu. Rev. Microbiol. 61:131–148.

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