INTRODUCTION

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The bacterium Vibrio cholerae causes the human diarrheal disease cholera. This organism colonizes the human small intestine, where it produces virulence factors that cause disease. Expression of a number of V. cholerae virulence factors, including the cholera toxin (CT) and the toxin coregulated pilus (TCP), is coordinately regulated by environmental signals resulting in high levels of expression within the host intestine but little to no expression outside the host (for a review, see reference 30). Coordinate expression of CT, TCP, and other virulence factors is controlled by a transmembrane DNA-binding protein, ToxR (27). ToxR requires another transmembrane transcriptional activator TcpP (13) to synergistically activate expression of toxT in response to specific laboratory conditions (14). ToxT is an AraC-like regulatory protein that directly activates transcription of several virulence genes, including ctx and tcp genes, which encode CT and TCP, respectively (8, 15).

ToxR, independent of TcpP and ToxT, also activates transcription of ompU (5), which encodes a major outer membrane porin (3) that has been suggested to be involved in adherence during pathogenesis (33). ToxR also represses the transcription of ompT, which encodes another outer membrane porin, in a TcpP- and ToxT-independent manner (V. DiRita, personal communication). These opposing activities of ToxR lead to virtually exclusive OmpU expression in wild-type strains and OmpT expression in toxR mutant strains, at least in vitro. Interestingly, both ToxR and OmpU homologues have been found in other Vibrio spp. (20, 29, 36), while toxT and the tcp and ctx genes are found on either a large pathogenicity island (17) or a lysogenic bacteriophage (35) only associated with epidemic V. cholerae strains. This suggests that toxT, tcp, and ctx genes were acquired relatively recently but toxR and ompU were present in the ancestral chromosome. The reasons for the ancestral regulatory protein ToxR gaining control over newly acquired virulence factor expression are unclear.

V. cholerae has the ability to cause global epidemics, or pandemics. It is believed that the first six cholera pandemics were caused by the classical V. cholerae biotype, while the seventh pandemic was caused by the El Tor biotype (1). These biotypes are differentiated in the laboratory by a number of characteristics, including different in vitro environmental signals which optimally induce virulence factor expression. Differential expression of virulence factors between the two biotypes of V. cholerae has been shown to be due to differential ToxR-dependent toxT expression (7). Presumably, ToxR-dependent transcription in both biotypes responds to common environmental signals within the host which have not yet been identified.

Bile is found at relatively high levels within the intestine, and resistance to bile is essential for enteric pathogens. Bile is composed primarily of bile salts, anionic detergents that not only aid in the digestion of fats but also are bacteriocidal due to their membrane solvent properties. The basic structure of the gram-negative bacteria provides some measure of resistance to bile by hiding the bile-sensitive cytoplasmic membrane beneath the relatively bile-resistant outer membrane (for a review, see reference 28). Lipopolysaccharide and outer membrane porins contribute to the resistance of Escherichia coli cells to bile (34). Additionally, efflux pumps have been identified which remove bile that reaches the cytoplasm of enteric bacteria (34). The inherent resistance of enteric bacteria to bile has been incorporated into their selective media, for example, thiosulfate-citrate-bile-sucrose (TCBS) for selection of Vibrio species. However, essentially nothing is known about V. cholerae resistance mechanisms to bile.

We demonstrate that ToxR mediates enhanced bile resistance in both biotypes of V. cholerae in a ToxT-independent manner in a way similar to the ToxR-dependent modulation of outer membrane porins. Moreover, ToxR modulates bile resistance and outer membrane protein expression in other pathogenic intestinal Vibrio species. Our results suggest that ToxR-dependent modulation of outer membrane proteins enhances bile resistance. Transcription of bile resistance gene(s) may have been one of the necessary prerequisites in the evolution of an ancestral transcriptional activator, ToxR, into the regulatory protein that controls virulence factor expression of an intestinal pathogen.

	MATERIALS AND METHODS

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Bacterial strains. E. coli DH5 (12) was used for cloning experiments, and strain SM10pir (26) was used to transfer plasmids to Vibrio strains by conjugation. All V. cholerae strains are isogenic with the classical strain O395 (23) or El Tor strain E7946 (24). V. cholerae KKV61, VJ740, and VJ739 have been described previously (4, 19). The toxR1 mutation was introduced into the chromosome of E7946 by plasmid pMD60, as previously described (19), to form strain KKV366. Strains O395, KKV61, VJ740, E7946, KKV366, and VJ739 were made phenotypically Lac for -galactosidase assays by the introduction of a chromosomal lacZ mutation with plasmid pCG711, as described previously (9), to form strains KKV598, KKV62, KKV163, KKV557, KKV555, and KKV556, respectively.

Plasmid construction. Amplification and cloning of toxR fragments using PCR with degenerate oligonucleotides has been described previously (29). These fragments were digested with SalI and EcoRI and then ligated into pGP704 (26) that had been similarly digested to form plasmids pKEK287 (V. cholerae toxR), pKEK266 (V. fluvialis toxR), pKEK260 (V. mimicus toxR), and pKEK273 (V. parahaemolyticus toxR). These plasmids were used to construct toxR Vibrio strains (see above).

Growth conditions and media. Minimum bactericidal concentration (MBC) and growth rate assays were performed by growth in Luria broth (LB) containing various concentrations of bile (sodium choleate; Sigma), deoxycholate (Amersham), sodium dodecyl sulfate (SDS; lauryl sulfate; Sigma), or Triton X-100 (Sigma). V. cholerae strains containing P_BAD vectors (e.g., in MBC and protein expression assays) were additionally grown in the presence of 0.05% arabinose and 50 µg of ampicillin per ml. V. cholerae strains were grown at 37°C, and the other Vibrio strains were grown at 30°C.

Transcription assays. V. cholerae strains containing plasmids pAL144 or pTL61T were grown (see above) to stationary phase in LB alone or supplemented with 0.4% bile or 0.1% DC; these conditions were chosen to match those used for the detection of protein expression. Media also contained 100 µg of ampicillin per ml for the retention of plasmid. Samples were permeabilized with chloroform and SDS and assayed for -galactosidase activity by the method of Miller (25).

Detection of protein expression. Outer membrane fractions were prepared as described previously (22, 26). Proteins were separated by SDS-10% polyacrylamide gel electrophoresis (PAGE) prior to Western blotting with rabbit polyclonal antisera against V. cholerae OmpU (6; the kind gift of J. Peterson) utilizing an ECL detection system (Amersham).

	RESULTS

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toxR V. cholerae strains of both biotypes have reduced MBCs of bile. The selective medium for Vibrio species, TCBS, contains a relatively high (0.8%) concentration of bile. We had observed a distinct growth defect of toxR V. cholerae strains relative to isogenic wild-type, toxT, or tcpP strains on this medium; the growth defect was not evident when the toxR strain was grown on the same medium lacking bile (data not shown). Based on this observation, experiments were designed to determine if ToxR plays a role in bile resistance of V. cholerae.

toxR mutant V. cholerae strains of both biotypes have reduced growth rates in the presence of anionic detergents. The MBCs for toxR strains of the anionic detergents (see above), while lower than those for isogenic wild-type strains, are still relatively high for these compounds, but the concentrations of individual bile salts within the intestine is probably lower. The bile salt concentration varies depending on the nutrition status, but it is estimated to be approximately 20 mM (~1%) within the small intestine, where V. cholerae colonizes (16). In order to determine if toxR strains exhibit defects at lower bile salt concentrations, growth rates were determined for both wild-type and toxR strains of both biotypes over a wide range of DC concentrations (Fig. 1A). The growth rate at each DC concentration relative to the growth rate in the absence of DC was plotted as a function of DC concentration. Although the toxR and wild-type strains have identical growth rates in the absence of DC, at every concentration of DC supplemented to the medium of >2 orders of magnitude, the toxR strains had slower growth rates than did the wild-type strains (Fig. 1A). While the toxR strains of both biotypes exhibited reduced growth rates in the presence of a wide range of DC, isogenic toxT strains had growth rates identical to those for the wild-type strains at all DC concentrations (data not shown).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   toxR mutant strains of classical and El Tor V. cholerae biotypes display slower growth rates in DC and SDS. (A) Relative growth rates of classical and El Tor V. cholerae strains in the presence of various concentrations of DC. Classical V. cholerae strains O395 (wt, ) and KKV61 (toxR, ) and El Tor V. cholerae strains E7946 (wt, ) and KKV366 (toxR, ) were grown in LB containing the DC concentrations indicated at 37°C (note the logarithmic scale for DC concentrations). Growth rates are shown relative to the growth rate in the absence of DC. (B) Relative growth rates of classical and El Tor V. cholerae strains in the presence of various concentrations of SDS. V. cholerae classical strains O395 (wt, ) and KKV61 (toxR, ) and El Tor strains E7946 (wt, ) and KKV366 (toxR, ) were grown in LB containing the SDS concentrations indicated at 37°C (note the logarithmic scale for SDS concentrations). Growth rates are shown relative to the growth rate in the absence of SDS.

Bile induces ToxR-dependent expression of OmpU in both biotypes. To identify ToxR-dependent factor(s) responsible for bile resistance that might be induced by the presence of bile or individual bile salts, wild-type and toxR strains of both biotypes were grown both in the absence or presence of bile and DC. Resolution of the total cellular proteins by SDS-PAGE revealed overexpression of a prominent ~38-kDa protein in wild-type strains of either biotype grown in the presence of bile or DC (Fig. 2). Overexpression of this protein made an absolute determination of the molecular weight difficult, but further fractionation experiments (see below) revealed that this protein was the apparent size of the ToxR-dependent outer membrane porin OmpU. The ~38-kDa protein was absent in toxR strains of either biotype grown either in the presence or in the absence of bile or DC; in these strains expression of the ToxR-repressed outer membrane porin OmpT (~40 kDa) is apparent. Total protein patterns of toxT strains or toxR strains carrying a plasmid expressing ToxR (pKEK86 or pKEK150) of either biotype were indistinguishable from those of the parental wild-type strains (not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 2.   A ToxR-dependent ~38-kDa protein is overexpressed during growth of classical and El Tor V. cholerae strains in bile and DC. Whole-cell lysates of V. cholerae classical strains (left panel) O395 (wild type) and KKV61 (toxR) and El Tor strains E7946 (wild type) and KKV366 (toxR) grown in LB alone, LB plus 0.4% bile, or LB plus 0.1% DC, as indicated above lanes. Samples were matched by equivalent OD600 units, separated by SDS-10% PAGE, and stained with Coomassie blue. The first lane of each panel has molecular mass markers, which are noted in kilodaltons to the left. The known mobilities of OmpU and OmpT are indicated by arrows.

View larger version (51K): [in this window] [in a new window]   FIG. 3.   The ToxR-dependent ~38-kDa protein localized in the outer membrane is OmpU. (A) Outer membrane fractions were prepared (22) of V. cholerae classical strains O395 (wt, lane 3) and KKV61 (toxR, lane 4) and El Tor strains E7946 (wt, lane 5) and KKV366 (toxR, lane 6) grown in LB with 0.4% bile (+bile); also shown are whole-cell lysates (total proteins) of strain O395 grown in LB alone (bile, lane 1) or LB plus 0.4% bile (+bile, lane 2). Samples were matched by equivalent OD600 units and were separated by SDS-10% PAGE and stained with Coomassie blue. The left lane has molecular mass markers, which are noted in kilodaltons. The known mobilities of OmpU and OmpT are indicated by arrows. The identity of the ~35-kDa outer membrane protein most apparent in toxR outer membranes is unknown. (B) The whole-cell and outer membrane protein samples above were subjected to Western analysis (see Materials and Methods) utilizing rabbit polyclonal antisera against OmpU.

ToxR-dependent transcription of ompU is stimulated by bile. Because OmpU is increased in the presence of bile, we tested whether this is due to increased ToxR-dependent ompU transcription. Wild-type, toxR, and toxT strains of both biotypes containing a plasmid with a ompU promoter-lacZ transcriptional fusion were measured for -galactosidase activity in the absence or the presence of bile and DC (Fig. 4). Only very low levels of ompU transcription were detected in toxR strains of either biotype under any growth condition, a finding consistent with the previous demonstration (5) that ToxR is required for high levels of ompU transcription. Relatively high levels of ompU transcription were detected in wild-type and toxT strains of both biotypes grown in LB alone. ompU transcription increased approximately two- to threefold when these strains were grown in the presence of either bile or DC. These results indicate that the overexpression of OmpU evident in toxR⁺ strains grown in bile or DC is apparently due at least in part to increased ToxR-dependent ompU transcription, suggesting that ToxR responds to bile.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   ToxR-dependent ompU transcription is increased in the presence of bile or DC. V. cholerae classical biotype strains KKV598 (wild type [wt]), KKV62 (toxR), and KKV163 (toxT), and El Tor biotype strains KKV557 (wt), KKV555 (toxR), and KKV556 (toxT) carrying the ompUp-lacZ transcriptional fusion plasmid pAL144 were grown in LB (open bars) alone or supplemented with 0.4% bile (shaded bars) or 0.1% DC (solid bars) and then assayed for -galactosidase as described in the text. Media also contained 100 µg of ampicillin per ml. Results are the average of three samples. The -galactosidase activity of each strain harboring the vector pTL61T (21) alone grown under these conditions (ca. 500 Miller U), which can be considered background activity, has been subtracted out.

ToxR modulates outer membrane proteins and enhanced bile resistance in V. mimicus, V. fluvialis, and V. parahaemolyticus. Because the toxR gene appears to be an ancestral Vibrio gene, we investigated whether ToxR-mediated outer membrane protein modulation and bile resistance were conserved among other Vibrio species and specifically in those that are intestinal pathogens. We previously identified toxR genes in two human intestinal pathogenic Vibrio species, V. mimicus and V. fluvialis (29), and toxR had been additionally identified in the intestinal pathogen V. parahaemolyticus (20). Insertional toxR mutant strains of V. mimicus and V. fluvialis, as well as of V. parahaemolyticus and V. cholerae were constructed as described (see Materials and Methods). The MBCs of bile, DC, and SDS were determined for wild-type and toxR mutant strains of V. mimicus, V. fluvialis, and V. parahaemolyticus. The toxR mutant strains of all three pathogenic Vibrio species exhibited lower MBCs for bile, DC, and SDS than the isogenic wild-type strains (Table 2).

View larger version (59K): [in this window] [in a new window]   FIG. 5.   Outer membrane proteins of V. mimicus, V. fluvialis, and V. parahaemolyticus are modulated by ToxR. (A) Outer membrane fractions were prepared as described earlier (26) of V. cholerae (V.c.) strains O395 (wild type [wt], lane 1) and O395toxR (toxR, lane 2), V. fluvialis (V.f.) strains 33809 (wt, lane 3) and 33809toxR (toxR, lane 4), V. mimicus (V.m.) strains 33655 (wt, lane 5) and 33655toxR (toxR, lane 6), and V. parahaemolyticus (V.p.) strains 43996 (wt, lane 7) and 43996toxR (toxR, lane 8). Strains were grown in LB supplemented with 0.4% bile. Samples were matched by equivalent OD600 units and separated by SDS-10% PAGE and stained with Coomassie blue. The mobility of molecular mass markers are noted in kilodaltons to the right. The known mobility of OmpU is indicated by arrow. (B) The outer membrane protein samples from wild-type and toxR mutant Vibrio strains (above) were subjected to Western analysis (see Materials and Methods) utilizing rabbit polyclonal antisera against OmpU; V. cholerae OmpU is indicated by an arrow.

	DISCUSSION

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The transmembrane protein ToxR is the master regulator of V. cholerae pathogenesis. ToxR is required for expression of the major virulence factors CT and TCP (27). However, ToxR is not the direct activator of TCP genes and apparently also not the direct activator of CT genes (4). Instead, ToxR, together with another transmembrane protein, TcpP, activates the toxT gene under inducing environmental conditions (8, 13). ToxT, an AraC-like transcriptional activator, then directly activates the genes encoding CT and TCP (4). The environmental signals that stimulate toxT transcription were originally thought to be sensed and responded to by ToxR (7) but now appear to be inducing conditions for the expression of tcpP (2, 31). Thus, TcpP, once made, appears to "coerce" ToxR into activating toxT transcription, something ToxR apparently does not normally do in the absence of TcpP (13). The tcp genes (including tcpP and toxT) are on a large pathogenicity island that may in fact be a filamentous bacteriophage (18), like bacteriophage CTX, which encodes the ctx genes (35). These horizontally transferable elements are found only in epidemic strains of V. cholerae, but toxR has been found in other bacteria within the genera Vibrio and Photobacterium (20, 29, 36). This leads to the question of what the original role of ToxR was in V. cholerae prior to acquisition of the cholera-specific virulence genes.

The original role of ToxR appears to be as a regulator of outer membrane proteins. In V. cholerae, ToxR, independent of ToxT and TcpP, activates transcription of ompU, which encodes a major outer membrane porin (5), and also represses transcription of ompT, which encodes another major outer membrane porin (V. DiRita, personal communication). In Photobacterium profundum ToxR likewise activates expression of a "porin-like" outer membrane protein OmpL while repressing expression of another outer membrane protein, OmpH (36). In this study we have demonstrated that ToxR modulates the expression of outer membrane proteins in V. mimicus, V. fluvialis, and V. parahaemolyticus, some of which are OmpU homologues (and therefore likely porins). These results suggest that the ancestral role of ToxR was as a modulator of outer membrane proteins, but then why would this protein be usurped as the regulator of virulence factor expression in V. cholerae?

The present study has uncovered a previously unknown role for ToxR as a modulator of enhanced bile resistance. ToxR, independent of ToxT, is required for enhanced survival and the growth of both V. cholerae biotypes in the presence of bile salts and anionic detergents. Moreover, ToxR is required for enhanced survival of the intestinal pathogens V. mimicus, V. fluvialis, and V. parahaemolyticus in the presence of bile salts and anionic detergents. All of these Vibrio species must be resistant to bile in order to persist within the intestine and cause disease. Perhaps this ancestral role in bile resistance led to the evolution of ToxR as the regulator of recently acquired virulence genes in V. cholerae that are expressed within the intestine.

Several lines of evidence suggest that the ToxR-regulated outer membrane proteins are involved in bile resistance. (i) OmpU and OmpT are the only known ToxR-dependent but ToxT- and TcpP-independent factors in V. cholerae, as with ToxR-dependent bile resistance. (ii) ToxR modulates both bile resistance and outer membrane proteins in other Vibrio species. (iii) OmpU is overexpressed when V. cholerae is grown in the presence of bile, apparently in part a result of increased ToxR-dependent ompU transcription. A previous study by Gupta and Chowdhury (10) failed to identify OmpU overexpression during V. cholerae growth in bile; in that study only outer membrane preparations were compared, rather than whole-cell lysates and ompU transcription, which may explain this discrepancy. One mechanism for ToxR-mediated enhanced bile resistance in V. cholerae would be inhibited influx of anionic detergents through the OmpU porin channel in comparison to the OmpT porin channel. This mechanism of bile resistance is seen in E. coli, where a strain expressing only the OmpF porin exhibits slower growth kinetics in the presence of DC compared to a strain expressing only the OmpC porin (34). However, no direct proof of OmpU and OmpT involvement in bile resistance exists yet in V. cholerae. An ompU mutant strain would be predicted to be more sensitive to bile, if OmpU has a protective role in the presence of bile, but we have not yet succeeded in our attempts to create such a mutant strain. Other laboratories have also noted failure in attempts to create a ompU V. cholerae strain, and this has been attributed to a possible essential role for OmpU (32).

ToxR transcribes high levels of ompU even in the absence of bile or DC, but in their presence ompU transcription increases, suggesting that ToxR transcriptional activity may be modulated by the presence of bile salts. The transcriptional activity of ToxR from P. profundum is modulated by pressure and also by local anesthetics such as procaine (36). Welch and Bartlett (36) postulate that both pressure and anesthetics change the membrane structure and that ToxR, which resides within the cytoplasmic membrane, actually responds to these membrane changes. Procaine has also been shown to modulate expression of the ToxR regulon in V. cholerae (13). Our studies indicate that ToxR may respond to another class of membrane-disruptive agents, namely, bile salts. A conserved mechanism of ToxR sensing and responding to membrane disruption in Vibrio species is an attractive hypothesis that awaits verification. The presence of bile salts in the environment signifies entry into the intestinal tract and would be a possible signal to stimulate ToxR-dependent transcription not only of bile resistance mechanisms but also of the ToxT-dependent virulence cascade.