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

The transmembrane regulatory protein ToxR is required for expression of vir ulence factors in the human diarrheal pathogen Vibrio cholerae, including c holera toxin (CT) and the toxin coregulated pilus (TCP), ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, w hich is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription o f the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic eleme nts, while the toxR, ompU, and ompT genes are apparently in the ancestral V ibrio chromosome. The contribution of ToxR-dependent modulation of outer me mbrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. I n both classical and El Tor biotypes of V. cholerae, a toxR mutant strain h as a reduced minimum bactericidal concentration (MBC) of bile, the bile com ponent 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 subin hibitory concentrations of bile or DC induces increased ToxR-dependent prod uction 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 i n both biotypes reveals stimulation (about two- to threefold) of ToxR-depen dent 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 addi tional 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 modulati on of outer membrane proteins and bile resistance of pathogenic Vibrio spec ies and suggest that these ToxR-dependent outer membrane proteins may media te enhanced resistance to bile. We speculate that ToxR-mediated bile resist ance was an early step in the evolution of V. cholerae as an intestinal pat hogen.