The virulence regulatory protein ToxR mediates enhanced bile resistance in Vibrio cholerae and other pathogenic Vibrio species

Abstract

The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, and ompT genes are apparently in the ancestral Vibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxT mutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. The toxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibrio species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution of V. cholerae as an intestinal pathogen.

FIG. 1

ΔtoxR 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 (ΔtoxR, ○) and El Tor V. cholerae strains E7946 (wt, ●) and KKV366 (ΔtoxR, ○) 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 (ΔtoxR, ○) and El Tor strains E7946 (wt, ●) and KKV366 (ΔtoxR, ○) 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.

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 (ΔtoxR) and El Tor strains E7946 (wild type) and KKV366 (ΔtoxR) 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.

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 (ΔtoxR, lane 4) and El Tor strains E7946 (wt, lane 5) and KKV366 (ΔtoxR, 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 ΔtoxR 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.

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 (ΔtoxR), and KKV163 (ΔtoxT), and El Tor biotype strains KKV557 (wt), KKV555 (ΔtoxR), and KKV556 (ΔtoxT) 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 β-galactosidase as described in the text. Media also contained 100 μg of ampicillin per ml. Results are the average of three samples. The β-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.

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.