Role of the Histone-Like Nucleoid Structuring Protein in Colonization, Motility, and Bile-Dependent Repression of Virulence Gene Expression
http://www.100md.com
感染与免疫杂志 2006年第5期
Biophysics Division, Indian Institute of Chemical Biology, Calcutta 700 032, India
ABSTRACT
Bile-mediated repression of virulence gene expression is relieved in a Vibrio cholerae hns mutant. The mutant also exhibited reduced motility due to lower flrA expression, higher in vivo production of the virulence factors, and lower colonization efficiency. The colonization defect of the mutant was due to low FlrA production.
TEXT
Vibrio cholerae, a gram-negative, noninvasive enteric bacterium is the causative agent of the diarrheal disease cholera (13). Cholera continues to be a cause of human mortality, especially in the developing countries, where conditions of poor sanitation, lack of safe drinking water, malnourishment, war, and famine contribute to regular episodes of cholera, estimated to be more than 5 million cases per year.
Expression of a subset of virulence factors of V. cholerae is controlled by a regulatory cascade known as the ToxR regulon (14). The transcriptional regulators ToxR and TcpP (8) act synergistically to control the production of a second regulator, ToxT (5), that directly activates the expression of several virulence genes, including ctxAB and tcpA, coding for cholera toxin (CT) and TcpA, the major subunit of the toxin-coregulated pilus, respectively. The toxin-coregulated pilus is thought to be essential for colonization of the intestinal epithelium by the bacterium (10, 27). Expression of the ToxR regulon is strongly influenced by physicochemical parameters that exert their effects at different levels of the regulatory cascade (reviewed in references 17, 20, and 26). Under nonpermissive conditions of temperature and pH, transcription of the tcpP gene is repressed, leading to down regulation of the entire virulence regulon (15). However, under anaerobic or microaerobic conditions, TcpP, ToxR, and ToxT are produced and the tcpA gene is optimally expressed, but expression of ctxAB is drastically reduced (16, 18). The presence of bile influences expression of the virulence regulon in yet another manner. Although production of the regulatory factors ToxR, TcpP, and ToxT is unaffected in cells grown in the presence of bile, expression of both ctxAB and tcpA is drastically reduced by an unknown mechanism (7, 24). A role of the nucleoid-associated histone-like nucleoid structuring protein (H-NS) in the silencing of virulence gene expression under nonpermissive conditions of temperature, pH, and oxygen concentration has been demonstrated (22, 28). In this study we have shown that H-NS also represses ctxAB and tcpA gene expression in cells grown in the presence of bile and in vivo in rabbit intestine. Furthermore, the motility and colonization efficiency of a V. cholerae hns mutant strain were lower than those of the wild-type strain.
A V. cholerae hns mutant produces CT and TcpA in the presence of bile. The V. cholerae O395 hns mutant strain O395H29, previously used to demonstrate the role of H-NS in anaerobic repression of CT production (16), was used in this study. CT in culture supernatants and sonicated cell lysates of V. cholerae strains O395 and O395H29 (hns) grown under inducing conditions (LB medium, pH 6.6, 30°C) without or with 0.2% bile (Sigma) was estimated by GM1 enzyme-linked immunosorbent assay. As reported previously (7), although V. cholerae O395 produced about 1 μg CT per ml supernatant per unit of optical density at 600 nm (OD600) under these conditions, practically no CT was detected in cultures of V. cholerae grown under identical conditions except for the presence of 0.2% bile in the culture medium. However, the hns mutant strain O395H29 produced high levels of CT even in the presence of bile (Fig. 1A). Taken together, these results indicated that H-NS represses CT production in the V. cholerae wild-type strain O395 grown in the presence of bile. When grown without bile, the hns mutant strain produced only about 20% more CT than the parent strain (P = 0.005) (Fig. 1A). Quantitative real-time reverse transcriptase PCR (RT-PCR) analysis (1) indicated that ctxAB expression was 19.4-fold lower in strain O395 than in strain O395H29 when both strains were grown in the presence of bile (Fig. 1B). Furthermore, the bile-mediated repression of tcpA expression reported for strain O395 was not observed in the hns mutant strain O395H29 (Fig. 1B).
Bile had no effect on toxT gene expression in both strains O395 and O395H29 (Fig. 1B). However, toxT expression was higher in the hns mutant strain O395H29 than in the parent strain O395 in both the absence and presence of bile. It has previously been shown that H-NS binding sites are present upstream of the toxT gene (22).
Paradoxically, although crude bile represses expression of the virulence factors, it has recently been demonstrated (12) that individual bile salts could actually stimulate ctxAB expression in a ToxR-dependent manner even under normally nonpermissive conditions (LB, pH 8.5, 37°C). It has been reported earlier that under these conditions significant CT production occurs in a V. cholerae hns mutant, although the parent strain produces practically no CT (22). Indeed, the V. cholerae hns mutant strain O395H29 used in this study produced about 1 μg CT per ml per OD600 unit when grown in LB at pH 8.5 and 37°C. The amount was further increased 1.5- to 2-fold when strain O395H29 was grown in the presence of the bile salt sodium glycocholate (Fig. 1A). To examine whether ToxR has any role in the sodium glycocholate-mediated increase in CT production in the hns mutant strain O395H29, a toxR hns double mutant of V. cholerae O395 was constructed by homologous integration of the suicide vector pGP704 (21), carrying an internal segment of the hns gene, into the chromosomal hns gene of the toxR mutant strain JJM43 (10). Southern blot analysis was used to confirm that integration had occurred at an appropriate position within the chromosomal hns gene. The toxR hns mutant strain was designated V. cholerae VCH3. Although the V. cholerae toxR mutant strain JJM43, which contained a functional hns gene, did not produce CT under any conditions, the toxR hns double mutant strain VCH3 produced constitutive levels of CT even under nonpermissive conditions (Fig. 1A). However, addition of sodium glycocholate had no effect on CT production in strain VCH3. These results suggested that even in the hns mutant strain, ToxR could, in the presence of the bile salt sodium glycocholate, modestly activate CT production. The activation was not observed in the hns toxR double mutant strain VCH3.
Since bile contains, in addition to bile acids, a variety of components such as bile pigments, lecithin, cholesterol, and probably some unidentified components, it is attractive to hypothesize that one or more of these components might directly or indirectly not only repress the activity of ToxT, as suggested earlier (24), but also prevent or decrease the bile acid-mediated ToxR-dependent stimulation of ctxAB expression observed by Hung and Mekalanos (12) and also in this study. Thus, even in the presence of significant amounts of bile acids in the bile used in the present and previous studies, no significant activation of ctxAB expression by ToxR was observed in strain O395 (7, 24).
Motility of the V. cholerae hns mutant. Expression of the ToxR virulence regulon and motility are known to be oppositely regulated in V. cholerae (3, 6). Indeed, while expression of the virulence genes ctxAB and tcpA of the ToxR regulon was increased in the V. cholerae hns mutant strain O395H29, swarm plate assays indicate that the motility of these cells was reduced compared to that of the wild-type parent strain O395 (Fig. 2A). To further understand the roles of the central virulence regulator ToxR and H-NS in motility, the motilities of strains O395, O395H29 (hns), JJM43 (toxR), and VCH3 (toxR hns) were compared under ToxR-inducing conditions (pH 6.6, 30°C) and ToxR-repressing conditions (pH 8.6, 37°C) by swarm plate assays (Fig. 2A). The results obtained indicate that in the presence of active ToxR (pH 6.6, 30°C), the swarm diameter of strain O395 (2.1 ± 0.3 cm) is twofold greater than the swarm diameter of the hns mutant strain O395H29 (1.1 ± 0.2 cm). The difference was statistically significant (P = 0.001) and suggested that H-NS positively regulates motility. However, when ToxR is inactive, either under nonpermissive growth conditions (pH 8.6, 37°C) or due to a mutation (strain JJM43), motility is increased twofold (P = 0.005), indicating that motility is negatively regulated by ToxR. In the toxR hns double mutant strain VCH3 motility is extremely high and comparable to that of the toxR mutant strain JJM43 (P 0.2), indicating that in the absence of an active negative regulator (ToxR), the positive regulator (H-NS) was not required.
Motility in V. cholerae is controlled by the hierarchical expression of a regulatory cascade (23). Real-time RT-PCR analysis indicated that expression of the first gene in the hierarchy, flrA, was reduced about sixfold (P = 0.001) in the hns mutant strain O395H29 (Fig. 2B). To examine whether the decrease in the motility of strain O395H29 was indeed due to reduced flrA expression, the full-length flrA gene was cloned in plasmid pBR328 to give plasmid pFlrA and transformed into strain O395H29. Expression of cloned FlrA in the V. cholerae hns mutant strain O395H29 restored motility to more than wild-type levels (Fig. 2A), indicating that H-NS exerts its effect on motility by stimulating expression of the central regulator flrA. RT-PCR analysis indicated that H-NS had no effect on expression of fliA (data not shown), encoding 54, which acts in concert with FlrA to activate expression of class II and class III motility genes (18).
In vivo expression of motility and virulence genes in the V. cholerae hns mutant. V. cholerae strains O395 and O395H29 (hns) were grown in vivo using the ligated rabbit ileal loop model (4). An advantage of this model is that it allows comparative studies between parent and mutant strains simultaneously in the same animal, thus avoiding variations among individual animals. Each bacterial strain was inoculated into at least two loops in each animal, and each strain was tested in at least three individual animals. At about 8 h after infection, the animals were sacrificed, bacteria were recovered from the loops and enumerated, and RNA was isolated from the in vivo-grown bacteria (3). The amount of CT in the intestinal fluid also was estimated. A statistically significant (P = 0.01) difference in CT production was observed between strains O395H29 (hns) and O395 grown in rabbit ileal loops. On a per-cell basis, strain O395H29 produced about 2.5-fold more CT in rabbit ileal loops than the parent strain O395 (Fig. 3B).
RT-PCR analysis of RNA isolated from in vivo-grown cells indicated that ctxAB expression was increased about 3.5-fold and tcpA expression was more than 2-fold higher in the hns mutant strain O395H29 compared to strain O395 (Fig. 3A). However, expression of the motility-regulatory gene flrA was about fivefold lower in strain O395H29 than in strain O395 (Fig. 3A). Thus, the effect of H-NS on gene expression in vivo closely follows the effect observed in in vitro cultures.
Decreased colonization of infant mouse intestine by the V. cholerae hns mutant. Colonization of infant mouse intestine by strain O395H29 was examined and compared to the colonization efficiency of the parent strain O395. Colonization by the mutant strain O395H29 was consistently about 20- to 30-fold lower than that by the wild-type strain (Table 1). Thus, in spite of the increased production of the colonization factor TcpA in the V. cholerae hns mutant strain, the colonization efficiency of the mutant was lower than that of the wild-type strain.
Whether the colonization defect of the hns mutant strain O395H29 was due to the lower motility of the mutant strain was next examined. Since expression of cloned flrA in the V. cholerae hns mutant strain O395H29 restored motility to wild-type levels (Fig. 2A), the colonization efficiency of strain O395H29 carrying pFlrA was examined in the infant mouse model and compared to that of the wild-type parent strain carrying the plasmid vector pBR328. The results obtained indicated that production of cloned FlrA in the hns mutant strain O395H29 fully complemented the colonization defect of the mutant strain (Table 1). Indeed, despite initial conflicting reports, it is now confirmed that motility is necessary for optimum colonization (2).
As colonization efficiency is also dependent on bacterial growth, the growth rates of strains O395 and O395H29 in LB were next determined and found to be comparable. Also, the ratios between the bacterial CFU injected into rabbit ileal loops and the CFU of bacteria recovered from the loops after 8 h were similar for strains O395 and O395H29 (data not shown).
A complex integrated regulatory system involving both positive and negative regulators not only controls virulence gene expression in response to external signals but also connects expression of virulence factors to motility, which are known to be oppositely regulated in V. cholerae. A model, incorporating the results of this study as well as previously reported observations, to depict the complex interplay between positive and negative regulators in controlling the expression of virulence factors and motility in response to environmental signals is given in Fig. 4. ToxR and ToxT function as positive regulators of the ctxAB and tcpA genes but directly or indirectly repress expression of the motility gene flrA. Conversely, H-NS is a negative regulator of ctxAB and tcpA gene expression but activates flrA expression. Consequently, expression of virulence factors and motility are oppositely regulated in V. cholerae. The regulators ToxR/ToxT and H-NS exert their activity under different environmental conditions. Under permissive conditions (LB, pH 6.6, 30°C), the ToxR/ToxT regulators predominate, with a consequent high level of expression of virulence factors but reduced motility. It may be mentioned in this context that the identity of the repressor of flrA expression is not precisely known; however, since induction of the ToxR regulon reduces motility, it may be hypothesized that ToxR directly or indirectly represses flrA expression. In the presence of bile, ToxT, though present, is not active, and H-NS predominantly represses expression of the virulence factors. H-NS also represses expression of the virulence factors under "nonpermissive" conditions (LB, pH 8.6, 37°C), where ToxT is not produced at all. The data presented in this study show that H-NS activates flrA expression, though H-NS is not required for flrA expression when the ToxR regulon is shut off. Since FlrA can fully complement the colonization defect of the V. cholerae hns mutant strain, it may be concluded that although H-NS is a pleiotropic regulator, its major role in the pathophysiology of infection is in activating flrA expression to achieve optimum colonization efficiency.
ACKNOWLEDGMENTS
We thank all members of the Biophysics Division for their cooperation, encouragement, and helpful discussions during this study and I. Guha Thakurtha and P. Majumdar for excellent technical support.
A.G. is grateful to the Council of Scientific and Industrial Research, Government of India, for a research fellowship. The work was supported by Network Project (SMM003) grants from the Council of Scientific and Industrial Research, Government of India.
REFERENCES
1. Ausbel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current protocols in molecular biology. John Wiley and Sons, New York, N.Y.
2. Butler, S. M., and A. Camilli. 2004. Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae. Proc. Natl. Acad. Sci. USA 101:5018-5023.
3. Chakraborty, S., N. Sengupta, and R. Chowdhury. 1999. Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae. Infect. Immun. 67:1025-1033.
4. De, S. N., and S. N. Chatterjee. 1953. An experimental study of the mechanisms of action of Vibrio cholerae on intestinal mucosal membrane. J. Pathol. Bacteriol. 46:559-562.
5. DiRita, V. J., C. Parsot, G. Jander, and J. J. Mekalanos. 1991. Regulatory cascade controls virulence in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 88:5403-5407.
6. Gardel, C. L., and J. J. Mekalanos. 1996. Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect. Immun. 64:2246-2255.
7. Gupta, S., and R. Chowdhury. 1997. Bile affects production of virulence factors and motility of Vibrio cholerae. Infect. Immun. 65:1131-1134.
8. Hase, C. C., and J. J. Mekalanos. 1998. TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 95:730-734.
9. Heidelberg, J. F., J. A. Eisen, W. C. Nelson, R. A. Clayton, M. L. Gwinn, R. J. Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, L. Umayam, S. R. Gill, K. E. Nelson, T. D. Read, H. Tettelin, D. Richardson, M. D. Ermolaeva, J. Vamathevan, S. Bass, H. Qin, I. Dragoi, P. Sellers, L. McDonald, T. Utterback, R. D. Fleishmann, W. C. Nierman, O. White, S. L. Salzberg, O. H. Smith, R. R. Colwell, J. J. Mekalanos, J. C. Venter, and C. M. Fraser. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477-483.
10. Herrington, D. A., R. H. Hall, G. Losonsky, J. J. Mekalanos, R. K. Taylor, and M. M. Levine. 1988. Toxin, toxin co-regulated pili and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med. 168:1487-1492.
11. Reference deleted.
12. Hung, D. T., and J. J. Mekalanos. 2005. Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner. Proc. Natl. Acad. Sci. USA 102:3028-3033.
13. Kaper, J. B., J. G. Morris, and M. M. Levine. 1995. Cholera. Clin. Microbiol. Rev. 8:48-86.
14. Klose, K. E. 2001. Regulation of virulence in Vibrio cholerae. Int. J. Med. Microbiol. 291:81-88.
15. Kovacikova, G., and K. Skorupski. 1999. A Vibrio cholerae LysR homolog, AphB, cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence cascade. J. Bacteriol. 181:4250-4256.
16. Krishnan, H. H., A. Ghosh, K. Paul, and R. Chowdhury. 2004. Effect of anaerobiosis on expression of virulence factors in Vibrio cholerae. Infect. Immun. 72:3961-3967.
17. Krokonis, E. S., and V. J. DiRita. 2003. From motility to virulence: sensing and responding to environmental signals in Vibrio cholerae. Curr. Opin. Microbiol. 6:186-190.
18. Lee, S. H., D. L. Hava, M. K. Waldor, and A. Camilli. 1999. Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99:625-634.
19. McCarter, L. L. 2001. Polar flagellar motility of Vibrionaceae. Microbiol. Mol. Biol. Rev. 65:445-462.
20. Mekalanos, J. J. 1992. Environmental signals controlling expression of virulence determinants in bacteria. J. Bacteriol. 174:1-7.
21. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in the construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-2583.
22. Nye, M. B., J. D. Pfau, K. Skorupski, and R. K. Taylor. 2000. Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade. J. Bacteriol. 182:4295-4303.
23. Prouty, M. G., N. E. Correa, and K. E. Klose. 2001. The novel sigma 54 and sigma 28-dependent flagellar gene transcription hierarchy of Vibrio cholerae. Mol. Microbiol. 39:1595-1609.
24. Schuhmacher, D. A., and K. K. Klose. 1999. Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae. J. Bacteriol. 181:1508-1514.
25. Sengupta, N., K. Paul, and R. Chowdhury. 2003. The global regulator ArcA modulates expression of virulence factors in Vibrio cholerae. Infect. Immun. 71:5583-5589.
26. Skorupski, K., and R. K. Taylor. 1997. Control of ToxR virulence regulon in Vibrio cholerae by environmental stimuli. Mol. Microbiol. 25:1003-1009.
27. Taylor, R. K., V. L. Miller, D. B. Furlong, and J. J. Mekalanos. 1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84:2833-2837.
28. Yu, R. R., and V. J. DiRita. 2002. Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation. Mol. Microbiol. 43:119-134.(Amalendu Ghosh, Kalidas P)
ABSTRACT
Bile-mediated repression of virulence gene expression is relieved in a Vibrio cholerae hns mutant. The mutant also exhibited reduced motility due to lower flrA expression, higher in vivo production of the virulence factors, and lower colonization efficiency. The colonization defect of the mutant was due to low FlrA production.
TEXT
Vibrio cholerae, a gram-negative, noninvasive enteric bacterium is the causative agent of the diarrheal disease cholera (13). Cholera continues to be a cause of human mortality, especially in the developing countries, where conditions of poor sanitation, lack of safe drinking water, malnourishment, war, and famine contribute to regular episodes of cholera, estimated to be more than 5 million cases per year.
Expression of a subset of virulence factors of V. cholerae is controlled by a regulatory cascade known as the ToxR regulon (14). The transcriptional regulators ToxR and TcpP (8) act synergistically to control the production of a second regulator, ToxT (5), that directly activates the expression of several virulence genes, including ctxAB and tcpA, coding for cholera toxin (CT) and TcpA, the major subunit of the toxin-coregulated pilus, respectively. The toxin-coregulated pilus is thought to be essential for colonization of the intestinal epithelium by the bacterium (10, 27). Expression of the ToxR regulon is strongly influenced by physicochemical parameters that exert their effects at different levels of the regulatory cascade (reviewed in references 17, 20, and 26). Under nonpermissive conditions of temperature and pH, transcription of the tcpP gene is repressed, leading to down regulation of the entire virulence regulon (15). However, under anaerobic or microaerobic conditions, TcpP, ToxR, and ToxT are produced and the tcpA gene is optimally expressed, but expression of ctxAB is drastically reduced (16, 18). The presence of bile influences expression of the virulence regulon in yet another manner. Although production of the regulatory factors ToxR, TcpP, and ToxT is unaffected in cells grown in the presence of bile, expression of both ctxAB and tcpA is drastically reduced by an unknown mechanism (7, 24). A role of the nucleoid-associated histone-like nucleoid structuring protein (H-NS) in the silencing of virulence gene expression under nonpermissive conditions of temperature, pH, and oxygen concentration has been demonstrated (22, 28). In this study we have shown that H-NS also represses ctxAB and tcpA gene expression in cells grown in the presence of bile and in vivo in rabbit intestine. Furthermore, the motility and colonization efficiency of a V. cholerae hns mutant strain were lower than those of the wild-type strain.
A V. cholerae hns mutant produces CT and TcpA in the presence of bile. The V. cholerae O395 hns mutant strain O395H29, previously used to demonstrate the role of H-NS in anaerobic repression of CT production (16), was used in this study. CT in culture supernatants and sonicated cell lysates of V. cholerae strains O395 and O395H29 (hns) grown under inducing conditions (LB medium, pH 6.6, 30°C) without or with 0.2% bile (Sigma) was estimated by GM1 enzyme-linked immunosorbent assay. As reported previously (7), although V. cholerae O395 produced about 1 μg CT per ml supernatant per unit of optical density at 600 nm (OD600) under these conditions, practically no CT was detected in cultures of V. cholerae grown under identical conditions except for the presence of 0.2% bile in the culture medium. However, the hns mutant strain O395H29 produced high levels of CT even in the presence of bile (Fig. 1A). Taken together, these results indicated that H-NS represses CT production in the V. cholerae wild-type strain O395 grown in the presence of bile. When grown without bile, the hns mutant strain produced only about 20% more CT than the parent strain (P = 0.005) (Fig. 1A). Quantitative real-time reverse transcriptase PCR (RT-PCR) analysis (1) indicated that ctxAB expression was 19.4-fold lower in strain O395 than in strain O395H29 when both strains were grown in the presence of bile (Fig. 1B). Furthermore, the bile-mediated repression of tcpA expression reported for strain O395 was not observed in the hns mutant strain O395H29 (Fig. 1B).
Bile had no effect on toxT gene expression in both strains O395 and O395H29 (Fig. 1B). However, toxT expression was higher in the hns mutant strain O395H29 than in the parent strain O395 in both the absence and presence of bile. It has previously been shown that H-NS binding sites are present upstream of the toxT gene (22).
Paradoxically, although crude bile represses expression of the virulence factors, it has recently been demonstrated (12) that individual bile salts could actually stimulate ctxAB expression in a ToxR-dependent manner even under normally nonpermissive conditions (LB, pH 8.5, 37°C). It has been reported earlier that under these conditions significant CT production occurs in a V. cholerae hns mutant, although the parent strain produces practically no CT (22). Indeed, the V. cholerae hns mutant strain O395H29 used in this study produced about 1 μg CT per ml per OD600 unit when grown in LB at pH 8.5 and 37°C. The amount was further increased 1.5- to 2-fold when strain O395H29 was grown in the presence of the bile salt sodium glycocholate (Fig. 1A). To examine whether ToxR has any role in the sodium glycocholate-mediated increase in CT production in the hns mutant strain O395H29, a toxR hns double mutant of V. cholerae O395 was constructed by homologous integration of the suicide vector pGP704 (21), carrying an internal segment of the hns gene, into the chromosomal hns gene of the toxR mutant strain JJM43 (10). Southern blot analysis was used to confirm that integration had occurred at an appropriate position within the chromosomal hns gene. The toxR hns mutant strain was designated V. cholerae VCH3. Although the V. cholerae toxR mutant strain JJM43, which contained a functional hns gene, did not produce CT under any conditions, the toxR hns double mutant strain VCH3 produced constitutive levels of CT even under nonpermissive conditions (Fig. 1A). However, addition of sodium glycocholate had no effect on CT production in strain VCH3. These results suggested that even in the hns mutant strain, ToxR could, in the presence of the bile salt sodium glycocholate, modestly activate CT production. The activation was not observed in the hns toxR double mutant strain VCH3.
Since bile contains, in addition to bile acids, a variety of components such as bile pigments, lecithin, cholesterol, and probably some unidentified components, it is attractive to hypothesize that one or more of these components might directly or indirectly not only repress the activity of ToxT, as suggested earlier (24), but also prevent or decrease the bile acid-mediated ToxR-dependent stimulation of ctxAB expression observed by Hung and Mekalanos (12) and also in this study. Thus, even in the presence of significant amounts of bile acids in the bile used in the present and previous studies, no significant activation of ctxAB expression by ToxR was observed in strain O395 (7, 24).
Motility of the V. cholerae hns mutant. Expression of the ToxR virulence regulon and motility are known to be oppositely regulated in V. cholerae (3, 6). Indeed, while expression of the virulence genes ctxAB and tcpA of the ToxR regulon was increased in the V. cholerae hns mutant strain O395H29, swarm plate assays indicate that the motility of these cells was reduced compared to that of the wild-type parent strain O395 (Fig. 2A). To further understand the roles of the central virulence regulator ToxR and H-NS in motility, the motilities of strains O395, O395H29 (hns), JJM43 (toxR), and VCH3 (toxR hns) were compared under ToxR-inducing conditions (pH 6.6, 30°C) and ToxR-repressing conditions (pH 8.6, 37°C) by swarm plate assays (Fig. 2A). The results obtained indicate that in the presence of active ToxR (pH 6.6, 30°C), the swarm diameter of strain O395 (2.1 ± 0.3 cm) is twofold greater than the swarm diameter of the hns mutant strain O395H29 (1.1 ± 0.2 cm). The difference was statistically significant (P = 0.001) and suggested that H-NS positively regulates motility. However, when ToxR is inactive, either under nonpermissive growth conditions (pH 8.6, 37°C) or due to a mutation (strain JJM43), motility is increased twofold (P = 0.005), indicating that motility is negatively regulated by ToxR. In the toxR hns double mutant strain VCH3 motility is extremely high and comparable to that of the toxR mutant strain JJM43 (P 0.2), indicating that in the absence of an active negative regulator (ToxR), the positive regulator (H-NS) was not required.
Motility in V. cholerae is controlled by the hierarchical expression of a regulatory cascade (23). Real-time RT-PCR analysis indicated that expression of the first gene in the hierarchy, flrA, was reduced about sixfold (P = 0.001) in the hns mutant strain O395H29 (Fig. 2B). To examine whether the decrease in the motility of strain O395H29 was indeed due to reduced flrA expression, the full-length flrA gene was cloned in plasmid pBR328 to give plasmid pFlrA and transformed into strain O395H29. Expression of cloned FlrA in the V. cholerae hns mutant strain O395H29 restored motility to more than wild-type levels (Fig. 2A), indicating that H-NS exerts its effect on motility by stimulating expression of the central regulator flrA. RT-PCR analysis indicated that H-NS had no effect on expression of fliA (data not shown), encoding 54, which acts in concert with FlrA to activate expression of class II and class III motility genes (18).
In vivo expression of motility and virulence genes in the V. cholerae hns mutant. V. cholerae strains O395 and O395H29 (hns) were grown in vivo using the ligated rabbit ileal loop model (4). An advantage of this model is that it allows comparative studies between parent and mutant strains simultaneously in the same animal, thus avoiding variations among individual animals. Each bacterial strain was inoculated into at least two loops in each animal, and each strain was tested in at least three individual animals. At about 8 h after infection, the animals were sacrificed, bacteria were recovered from the loops and enumerated, and RNA was isolated from the in vivo-grown bacteria (3). The amount of CT in the intestinal fluid also was estimated. A statistically significant (P = 0.01) difference in CT production was observed between strains O395H29 (hns) and O395 grown in rabbit ileal loops. On a per-cell basis, strain O395H29 produced about 2.5-fold more CT in rabbit ileal loops than the parent strain O395 (Fig. 3B).
RT-PCR analysis of RNA isolated from in vivo-grown cells indicated that ctxAB expression was increased about 3.5-fold and tcpA expression was more than 2-fold higher in the hns mutant strain O395H29 compared to strain O395 (Fig. 3A). However, expression of the motility-regulatory gene flrA was about fivefold lower in strain O395H29 than in strain O395 (Fig. 3A). Thus, the effect of H-NS on gene expression in vivo closely follows the effect observed in in vitro cultures.
Decreased colonization of infant mouse intestine by the V. cholerae hns mutant. Colonization of infant mouse intestine by strain O395H29 was examined and compared to the colonization efficiency of the parent strain O395. Colonization by the mutant strain O395H29 was consistently about 20- to 30-fold lower than that by the wild-type strain (Table 1). Thus, in spite of the increased production of the colonization factor TcpA in the V. cholerae hns mutant strain, the colonization efficiency of the mutant was lower than that of the wild-type strain.
Whether the colonization defect of the hns mutant strain O395H29 was due to the lower motility of the mutant strain was next examined. Since expression of cloned flrA in the V. cholerae hns mutant strain O395H29 restored motility to wild-type levels (Fig. 2A), the colonization efficiency of strain O395H29 carrying pFlrA was examined in the infant mouse model and compared to that of the wild-type parent strain carrying the plasmid vector pBR328. The results obtained indicated that production of cloned FlrA in the hns mutant strain O395H29 fully complemented the colonization defect of the mutant strain (Table 1). Indeed, despite initial conflicting reports, it is now confirmed that motility is necessary for optimum colonization (2).
As colonization efficiency is also dependent on bacterial growth, the growth rates of strains O395 and O395H29 in LB were next determined and found to be comparable. Also, the ratios between the bacterial CFU injected into rabbit ileal loops and the CFU of bacteria recovered from the loops after 8 h were similar for strains O395 and O395H29 (data not shown).
A complex integrated regulatory system involving both positive and negative regulators not only controls virulence gene expression in response to external signals but also connects expression of virulence factors to motility, which are known to be oppositely regulated in V. cholerae. A model, incorporating the results of this study as well as previously reported observations, to depict the complex interplay between positive and negative regulators in controlling the expression of virulence factors and motility in response to environmental signals is given in Fig. 4. ToxR and ToxT function as positive regulators of the ctxAB and tcpA genes but directly or indirectly repress expression of the motility gene flrA. Conversely, H-NS is a negative regulator of ctxAB and tcpA gene expression but activates flrA expression. Consequently, expression of virulence factors and motility are oppositely regulated in V. cholerae. The regulators ToxR/ToxT and H-NS exert their activity under different environmental conditions. Under permissive conditions (LB, pH 6.6, 30°C), the ToxR/ToxT regulators predominate, with a consequent high level of expression of virulence factors but reduced motility. It may be mentioned in this context that the identity of the repressor of flrA expression is not precisely known; however, since induction of the ToxR regulon reduces motility, it may be hypothesized that ToxR directly or indirectly represses flrA expression. In the presence of bile, ToxT, though present, is not active, and H-NS predominantly represses expression of the virulence factors. H-NS also represses expression of the virulence factors under "nonpermissive" conditions (LB, pH 8.6, 37°C), where ToxT is not produced at all. The data presented in this study show that H-NS activates flrA expression, though H-NS is not required for flrA expression when the ToxR regulon is shut off. Since FlrA can fully complement the colonization defect of the V. cholerae hns mutant strain, it may be concluded that although H-NS is a pleiotropic regulator, its major role in the pathophysiology of infection is in activating flrA expression to achieve optimum colonization efficiency.
ACKNOWLEDGMENTS
We thank all members of the Biophysics Division for their cooperation, encouragement, and helpful discussions during this study and I. Guha Thakurtha and P. Majumdar for excellent technical support.
A.G. is grateful to the Council of Scientific and Industrial Research, Government of India, for a research fellowship. The work was supported by Network Project (SMM003) grants from the Council of Scientific and Industrial Research, Government of India.
REFERENCES
1. Ausbel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current protocols in molecular biology. John Wiley and Sons, New York, N.Y.
2. Butler, S. M., and A. Camilli. 2004. Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae. Proc. Natl. Acad. Sci. USA 101:5018-5023.
3. Chakraborty, S., N. Sengupta, and R. Chowdhury. 1999. Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae. Infect. Immun. 67:1025-1033.
4. De, S. N., and S. N. Chatterjee. 1953. An experimental study of the mechanisms of action of Vibrio cholerae on intestinal mucosal membrane. J. Pathol. Bacteriol. 46:559-562.
5. DiRita, V. J., C. Parsot, G. Jander, and J. J. Mekalanos. 1991. Regulatory cascade controls virulence in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 88:5403-5407.
6. Gardel, C. L., and J. J. Mekalanos. 1996. Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect. Immun. 64:2246-2255.
7. Gupta, S., and R. Chowdhury. 1997. Bile affects production of virulence factors and motility of Vibrio cholerae. Infect. Immun. 65:1131-1134.
8. Hase, C. C., and J. J. Mekalanos. 1998. TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 95:730-734.
9. Heidelberg, J. F., J. A. Eisen, W. C. Nelson, R. A. Clayton, M. L. Gwinn, R. J. Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, L. Umayam, S. R. Gill, K. E. Nelson, T. D. Read, H. Tettelin, D. Richardson, M. D. Ermolaeva, J. Vamathevan, S. Bass, H. Qin, I. Dragoi, P. Sellers, L. McDonald, T. Utterback, R. D. Fleishmann, W. C. Nierman, O. White, S. L. Salzberg, O. H. Smith, R. R. Colwell, J. J. Mekalanos, J. C. Venter, and C. M. Fraser. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477-483.
10. Herrington, D. A., R. H. Hall, G. Losonsky, J. J. Mekalanos, R. K. Taylor, and M. M. Levine. 1988. Toxin, toxin co-regulated pili and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med. 168:1487-1492.
11. Reference deleted.
12. Hung, D. T., and J. J. Mekalanos. 2005. Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner. Proc. Natl. Acad. Sci. USA 102:3028-3033.
13. Kaper, J. B., J. G. Morris, and M. M. Levine. 1995. Cholera. Clin. Microbiol. Rev. 8:48-86.
14. Klose, K. E. 2001. Regulation of virulence in Vibrio cholerae. Int. J. Med. Microbiol. 291:81-88.
15. Kovacikova, G., and K. Skorupski. 1999. A Vibrio cholerae LysR homolog, AphB, cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence cascade. J. Bacteriol. 181:4250-4256.
16. Krishnan, H. H., A. Ghosh, K. Paul, and R. Chowdhury. 2004. Effect of anaerobiosis on expression of virulence factors in Vibrio cholerae. Infect. Immun. 72:3961-3967.
17. Krokonis, E. S., and V. J. DiRita. 2003. From motility to virulence: sensing and responding to environmental signals in Vibrio cholerae. Curr. Opin. Microbiol. 6:186-190.
18. Lee, S. H., D. L. Hava, M. K. Waldor, and A. Camilli. 1999. Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99:625-634.
19. McCarter, L. L. 2001. Polar flagellar motility of Vibrionaceae. Microbiol. Mol. Biol. Rev. 65:445-462.
20. Mekalanos, J. J. 1992. Environmental signals controlling expression of virulence determinants in bacteria. J. Bacteriol. 174:1-7.
21. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in the construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-2583.
22. Nye, M. B., J. D. Pfau, K. Skorupski, and R. K. Taylor. 2000. Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade. J. Bacteriol. 182:4295-4303.
23. Prouty, M. G., N. E. Correa, and K. E. Klose. 2001. The novel sigma 54 and sigma 28-dependent flagellar gene transcription hierarchy of Vibrio cholerae. Mol. Microbiol. 39:1595-1609.
24. Schuhmacher, D. A., and K. K. Klose. 1999. Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae. J. Bacteriol. 181:1508-1514.
25. Sengupta, N., K. Paul, and R. Chowdhury. 2003. The global regulator ArcA modulates expression of virulence factors in Vibrio cholerae. Infect. Immun. 71:5583-5589.
26. Skorupski, K., and R. K. Taylor. 1997. Control of ToxR virulence regulon in Vibrio cholerae by environmental stimuli. Mol. Microbiol. 25:1003-1009.
27. Taylor, R. K., V. L. Miller, D. B. Furlong, and J. J. Mekalanos. 1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84:2833-2837.
28. Yu, R. R., and V. J. DiRita. 2002. Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation. Mol. Microbiol. 43:119-134.(Amalendu Ghosh, Kalidas P)