Infectious Diseases, St Jude Childrens Research Hospital, 332 N Lauderdale Street, Memphis, TN 38105, USA1
Tel: +1 901 495 2865. Fax: +1 901 495 3099. e-mail: claudia.hase{at}stjude.org
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ABSTRACT |
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Keywords: cholera, mechanosensitive ion channels, motility, sodium bioenergetics
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; CT, cholera toxin; MS, mechanosensitive; PVP, polyvinylpyrrolidone; s.m.f., sodium motive force; TCP, toxin-coregulated pili
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INTRODUCTION |
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Motility is an important virulence factor in many pathogenic species and in some cases is inversely regulated with the expression of virulence traits (Ottemann & Miller, 1997 ). Although the role of motility of V. cholerae in its ability to cause cholera has not been clearly established, the production of CT and TCP are known to be affected by the motility phenotype of the bacteria. At least two ToxR-regulated genes on the TCP-ACF island, tcpI and acfB, encode proteins with homology to chemoreceptors, and mutations in these two genes positively affect motility of V. cholerae as assayed by swarm plate assays (Everiss et al., 1994
; Harkey et al., 1994
). Furthermore, toxR mutant strains display a hypermotile phenotype, whereas some spontaneous hypermotile mutants lack expression of CT and TCP under normally inducing conditions (Gardel & Mekalanos, 1996
). Conversely, some non-motile mutants show constitutive expression of CT and TCP under alkaline conditions and increased toxT transcription (Gardel & Mekalanos, 1996
; Häse & Mekalanos, 1999
).
Many bacterial species respond to changes in the viscosity of their surrounding environment in various ways. In some cases, exposure to a semi-solid surface results in morphological changes that allow the bacteria to spread along the surface and to form biofilms. For example, exposure of Vibrio parahaemolyticus to a semi-solid surface or increasing media viscosity results in the induction of laf genes, encoding proteins required for the production of lateral flagella that allow the bacteria to swarm (McCarter, 1999 ). As increasing the drag on the single polar flagellum by either antibodies or increased media viscosity induced laf genes, it was suggested that the polar flagellum might act as a mechanosensor (Kawagishi et al., 1996
). However, the mechanism by which the bacteria sense this change in the environment is not yet understood. It was suggested that the bacteria might sense a decrease in the rotation rate of, or the sodium influx through, the polar flagellar motor (Kawagishi et al., 1996
).
V. cholerae experiences a high-viscosity environment in the mucus lining of the gut. It was recently reported that increases in media viscosity result in increased toxT::lacZ expression (Häse & Mekalanos, 1999 ) and perhaps the ability of the organism to sense the viscosity of the environment, possibly by monitoring flagellar activity, is an important step for the induction of virulence gene expression. It was suggested that the sodium flux through the flagellum is sensed by the bacteria and might be an important regulatory signal, resulting in altered virulence gene expression in V. cholerae (Häse & Mekalanos, 1999
). Changing the bacterial membrane bioenergetics by mutation of a primary sodium pump (NQR), or by the addition of various drugs that negatively affect motility, also resulted in altered virulence gene expression (Häse & Mekalanos, 1999
). Thus, the V. cholerae flagellum might be a sensitive voltmeter, responding to very small changes in membrane potential (Dibrov, 2000
).
In the present study, the role of the V. cholerae single polar flagellum in virulence gene regulation was investigated.
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METHODS |
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Genetic manipulations.
Mutants of V. cholerae were generated by homologous recombination. Preliminary sequence data for V. cholerae were obtained from The Institute for Genomic Research website at http://www.tigr.org. The genes and surrounding sequences were amplified in PCR reactions by using specific primers and cloned into the plasmid vectors pCR2.1 (Invitrogen) or pUC19. Internal deletions in yggB and mscL were generated by using convenient restriction sites present in the genes, and the DNA was then subcloned into pWM91 (Metcalf et al., 1996 ) (generously provided by B. Wanner). The mutated alleles were introduced into the chromosome of the O395N1 toxT::lacZ strain following sucrose selection as described by Donnenberg & Kaper (1991)
. Plasmid DNA was prepared by using the Qiagen Miniprep extraction kit (Chatsworth) and introduced into bacteria by electroporation.
Biochemical assays.
ß-Galactosidase activities were assayed as described by Häse & Mekalanos (1999) and Miller (1972)
.
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RESULTS |
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Effects on TcpP/H
A toxT::lacZ reporter strain carrying deletions in the toxR and tcpP genes complemented by a plasmid with arabinose-inducible tcpPH genes has previously been shown to show high levels of ß-galactosidase in the presence of the inducer (Häse & Mekalanos, 1998 ). Fig. 5
shows the effects of various growth conditions or drugs demonstrated above to affect toxT::lacZ expression on the ß-galactosidase levels of this strain. When the TcpP/H proteins are functioning in the absence of ToxR/S to activate the toxT::lacZ reporter construct, similar trends in the effects of several different conditions on ß-galactosidase expression were observed in this strain compared to the parental strain (Fig. 5
), indicating that the ToxR/S proteins are not required for the sensing of these conditions.
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DISCUSSION |
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Mechanosensation is ubiquitous in living cells. In E. coli, three distinct MS ion channel activities can be observed by the patch-clamp technique (Berrier et al., 1996 ; Martinac et al., 1987
). To date, only two genes, mscL and yggB, encoding MS channel activities have been cloned (Levina et al., 1999
; Sukharev et al., 1994
). As the flagellum did not appear to be involved in the sensing of viscosity, we hypothesized that perhaps MS channels are required for the ability of the bacteria to recognize changes in the viscosity of the surrounding media. It was previously noted that the addition of procaine, a membrane-intercalating amphipathic drug known to activate MS ion channels in patch-clamp experiments (Martinac et al., 1990
), strongly reduced expression of TCP in V. cholerae (Häse & Mekalanos, 1999
). Consistent with a possible role of MS channels in virulence gene regulation in V. cholerae, procaine decreased toxT::lacZ transcription, whereas the addition of gadolinium, a specific inhibitor of MS channel activity (Berrier et al., 1992
; Häse et al., 1997
), increased toxT::lacZ levels. A mutant derivative strain carrying deletions in the V. cholerae mscL and yggB homologues was constructed and found to be sensitive to osmotic down-shock similar to that observed in the E. coli double mutant (Levina et al., 1999
). However, like the parent strain, the mscL yggB double mutant derivative strain showed increased toxT::lacZ expression in high-viscosity media, indicating that these two putative ion channels are not required in this response. Interestingly, no significant difference in toxT::lacZ expression levels even in the presence of procaine or gadolinium was found between the double mutant and parental strains, indicating that the effects of these drugs are mediated independently of the putative MscL and MscS channels. The lack of effects of these mutations is currently not understood; however, it is likely that V. cholerae also has the third, as yet uncloned, type of MS channel found in E. coli, MscM, and perhaps the presence of this channel masks any effects of the other mutations. Procaine is known to affect the membrane fluidity and osmotic regulation of porin genes in E. coli (Rampersaud & Inouye, 1991
; Tanji et al., 1992
), and perhaps its effects on virulence gene regulation in V. cholerae might be the consequence of a more general effect on the physio-chemical state of the membrane. Further experiments are required to better understand these results.
The expression of CT and TCP in V. cholerae is strongly influenced by changes in growth conditions, at least in laboratory media (Skorupski & Taylor, 1997 ). The mechanisms of the signal transduction events leading to the repression of these virulence factors are not yet understood. As ToxR/S and TcpP/H are integral membrane proteins, they might be able to directly respond to changes in the surrounding media, perhaps by changing their conformation and/or dimerization. The generation of a toxT::lacZ reporter strain allowed us a quantitative analysis of several non-inducing conditions on the expression levels of the toxT gene. It was previously shown that toxT transcription in classical biotype V. cholerae is reduced under alkaline conditions and at high NaCl levels (DiRita et al., 1991
; Häse & Mekalanos, 1999
). Although growth of classical biotype V. cholerae at 37 °C results in dramatic repression of CT and TCP production (Miller & Mekalanos, 1988
), the transcription of the toxT gene was not very strongly reduced at high temperature, consistent with the recent finding that temperature negatively affects ToxT transcriptional activity (Schumacher & Klose, 1999
). We now have a powerful tool to dissect the levels in this regulatory cascade at which various non-inducing conditions exert their effects.
Some of the environmental conditions that negatively affect the ToxR regulon, such as bile and pH, are known to positively affect motility (Gupta & Chowdhury, 1997 ; Kiiyukia et al., 1993
). The previously established connection between the motility phenotype of the bacteria and the expression of ToxR-regulated genes led to the model that perhaps some of the signals affecting the ToxR regulon do so by altering motility (Häse & Mekalanos, 1999
). A prediction from this model would be that a non-motile mutant strain should be blind to these repressing conditions. However, toxT transcription of a mutant derivative deleted in the fliG gene, thus producing no flagella, was affected by various environmental conditions similarly to the motile parent strain, indicating that the flagellum does not play a major role in the signal transduction events, at least for the tested conditions.
We recently demonstrated that mutations in the nqr gene cluster, encoding a primary sodium extrusion pump, of V. cholerae resulted in increased virulence factor expression (Häse & Mekalanos, 1998 , 1999
). It is conceivable that the observed effects of the nqr mutation on toxT transcription are mediated indirectly via motility. Unlike E. coli, the single polar flagellum of V. cholerae is energized by the s.m.f. via translocation of sodium ions (Häse & Mekalanos, 1999
; Kojima et al., 1999
). As the activity of the NQR enzyme is believed to generate a s.m.f. that can energize flagellar rotation, a lack of NQR activity may reduce s.m.f., which in turn may slow the flagella. Consistent with this idea, inhibition of flagellar rotation by the addition of phenamil (an amiloride homologue that is a known inhibitor of sodium-driven flagellar motors) or monensin (an ionophore that changes the level of Na+ chemical potential), or introduction of mutations resulting in non-motile phenotypes, resulted in increases in toxT::lacZ expression and CT production under in vitro expression conditions (Gardel & Mekalanos, 1996
; Häse & Mekalanos, 1999
). These observations led to the hypothesis that perhaps Na+ flux through the flagellum might directly be a signal for gene regulation. Here, it is shown that the protonophore CCCP alone and in combination with monensin increased toxT::lacZ expression, suggesting that the cells can indeed monitor the membrane potential (
), as suggested previously by Dibrov (2000)
. However, the non-flagellate mutant derivative strain responded to the addition of the ionophores and of phenamil similarly to the motile parent strain. It should be noted that amiloride and its analogues inhibit the bacterial antiporter NhaB (Pinner et al., 1995
), thus potentially affecting the overall membrane bioenergetics. Moreover, elevated toxT::lacZ expression was observed in strains carrying a nqr mutation in combination with motility defects. Together, these results indicate that the flagellum is not required for this sensing of changes in the membrane energy levels on toxT transcription. Given the supposed tight coupling of flagellar motors, a drop in s.m.f. would probably affect other s.m.f.-dependent systems before any change in flagellar rotation occurs. However, the monitoring of
by an as yet uncharacterized mechanism might be very important and may provide V. cholerae with the necessary clues that convert this organism from its environmental to its pathogenic phase.
To address the individual roles of the ToxR/S and TcpP/H proteins, if any, in the sensing of environmental changes, a reporter strain in which the activation of the toxT gene is achieved by overexpression of the TcpP/H proteins in the absence of the ToxR/S proteins (Häse & Mekalanos, 1998 ) was used to analyse the effects of the various growth conditions and compounds shown above to alter toxT::lacZ expression. Thus, if ToxR/S are necessary for the sensing of media viscosity this strain should lack the ability to respond to this condition. Interestingly, the relative activation levels of the reporter construct achieved by the TcpP/H molecules alone were somewhat comparable to those observed in the wild-type strain, suggesting that the ToxR/S proteins do not play a major role in the sensing of these signals.
Although the very attractive and intriguing hypothesis of flagella-mediated sensing of environmental changes was shown to be incorrect in this study, it is important to note that there must be alternative systems for the sensing of some of these conditions in V. cholerae. Analysing the underlying mechanisms by which bacteria respond to changes in the environment, such as their ability to monitor the level of membrane potential, will probably reveal complex interplays between basic physiological processes and virulence factor expression in a variety of pathogenic species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Berrier, C., Besnard, M., Ajouz, B., Coulombe, A. & Ghazi, A. (1996). Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure. J Membr Biol 151, 175-187.[Medline]
Dibrov, P. (2000). Lowering the electric potential on the membrane as a possible signal modulating the expression of virulence factors in Vibrio cholerae. Mol Microbiol 35, 473-475.[Medline]
DiRita, V. J. (1995). Three-component regulatory system controlling virulence in Vibrio cholerae. In Two-Component Signal Transduction , pp. 351-365. Edited by J. A. Hoch & T. J. Silhavy. Washington, DC: American Society for Microbiology.
DiRita, V. J., Parsot, C., Jander, G. & Mekalanos, J. J. (1991). Regulatory cascade controls virulence in Vibrio cholerae. Proc Natl Acad Sci USA 88, 5403-5407.[Abstract]
Donnenberg, M. S. & Kaper, J. B. (1991). Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59, 4310-4317.[Medline]
Everiss, K. D., Hughes, K. J., Kovach, M. E. & Peterson, K. M. (1994). The Vibrio cholerae acfB colonization determinant encodes an inner membrane protein that is related to a family of signal-transducing proteins. Infect Immun 62, 3289-3298.[Abstract]
Gardel, C. & Mekalanos, J. J. (1996). Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect Immun 64, 2246-2255.[Abstract]
Gosink, K. K. & Häse, C. C. (2000). Requirements for the conversion of the Na+-driven flagellar motor of Vibrio cholerae to the H+-driven motor of Escherichia coli. J Bacteriol 182, 4234-4240.
Gupta, S. & Chowdhury, R. (1997). Bile affects production of virulence factors and motility of Vibrio cholerae. Infect Immun 65, 1131-1134.[Abstract]
Harkey, C. W., Everiss, K. D. & Peterson, K. M. (1994). The Vibrio cholerae toxin-coregulated-pilus gene tcpI encodes a homolog of methyl-accepting chemotaxis proteins. Infect Immun 62, 2669-2678.[Abstract]
Häse, C. C. & Mekalanos, J. J. (1998). TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 95, 730-734.
Häse, C. C. & Mekalanos, J. J. (1999). Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 96, 3183-3187.
Häse, C. C., LeDain, A. C. & Martinac, B. (1997). Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli mutants with altered channel gating and pressure sensitivity. J Membr Biol 157, 17-25.[Medline]
Holmgren, J. & Svennerholm, A. M. (1973). Enzyme-linked immunosorbent assays for cholera serology. Infect Immun 7, 759-763.[Medline]
Kawagishi, I., Imagawa, M., Imae, Y., McCarter, L. & Homma, M. (1996). The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar gene expression. Mol Microbiol 20, 693-699.[Medline]
Kiiyukia, C., Kawakami, H. & Hashimoto, H. (1993). Effect of sodium chloride, pH and organic nutrients on the motility of Vibrio cholerae non-01. Microbios 73, 249-255.[Medline]
Klose, K. E. & Mekalanos, J. J. (1998). Differential regulation of multiple flagellins in Vibrio cholerae. J Bacteriol 180, 303-316.
Kojima, S. K. Y., Kawagishi, I. & Homma, M. (1999). The polar flagellar motor of Vibrio cholerae is driven by a Na+ motive force. J Bacteriol 181, 1927-1930.
Levina, N., Totemeyer, S., Stokes, N. R., Louis, P., Jones, M. A. & Booth, I. R. (1999). Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J 18, 1730-1737.
McCarter, L. (1999). The multiple identities of Vibrio parahaemolyticus. J Mol Microbiol Biotechnol 1, 51-57.[Medline]
Martinac, B., Buechner, M., Delcour, A., Adler, J. & Kung, C. (1987). Pressure-sensitive ion channels in Escherichia coli. Proc Natl Acad Sci USA 84, 2297-2301.[Abstract]
Martinac, B., Adler, J. & Kung, C. (1990). Mechanosensitive ion channels of E. coli activated by amphipaths. Nature 348, 261-263.[Medline]
Metcalf, W. W., Jiang, W., Daniels, L. L., Kim, S. K., Haldimann, A. & Wanner, B. L. (1996). Conditionally replicative and conjugative plasmids carrying lacZ for cloning, mutagenesis, and allelle replacement in bacteria. Plasmid 35, 1-13.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Miller, V. & Mekalanos, J. (1988). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170, 2575-2583.[Medline]
Ottemann, K. M. & Miller, J. F. (1997). Roles for motility in bacterialhost interactions. Mol Microbiol 24, 1109-1117.[Medline]
Pinner, E., Padan, E. & Schuldiner, S. (1995). Amiloride and harmaline are potent inhibitors of NhaB, a Na+/H+ antiporter from Escherichia coli. FEBS Lett 365, 18-22.[Medline]
Rampersaud, A. & Inouye, M. (1991). Procaine, a local anesthetic, signals through the EnvZ receptor to change the DNA binding affinity of the transcriptional activator protein OmpR. J Bacteriol 173, 6882-6888.[Medline]
Schumacher, D. & Klose, K. (1999). Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae. J Bacteriol 181, 1508-1514.
Skorupski, K. & Taylor, R. K. (1997). Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli. Mol Microbiol 25, 1003-1009.[Medline]
Sukharev, S. I., Blount, P., Martinac, B., Blattner, F. R. & Kung, C. (1994). A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265-268.[Medline]
Tanji, K., Ohta, Y., Kawato, S., Mizushima, T., Natori, S. & Sekimizu, K. (1992). Decrease of psychotropic drugs and local anaesthetics of membrane fluidity measured by fluorescence anisotropy in Escherichia coli. J Pharm Pharmacol 44, 1036-1037.[Medline]
Received 3 November 2000;
revised 13 December 2000;
accepted 15 December 2000.