Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus

Abstract

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.

Figure 1. CT and TCP production by RND mutants.

CT and TCP production in the indicated strains was determined following growth under AKI conditions. CT (A) and TcpA (B) were detected by CT GM₁-ELISA and TcpA Western immunoblotting, respectively. Error bars represent the standard deviation of the mean from three or more experiments. Statistical analysis was performed by one-way ANOVA. *p<0.05 compared to wild-type (WT); **p<0.05 compared to all tested strains.

Figure 2. Infant mouse colonization assays with the RND efflux mutants.

Competition assays were performed using the infant mouse colonization assay as described in the Materials and Methods. Infant mice were challenged with a ∼10⁵ cfu inoculum containing a mixture of wild-type and the indicated mutant at a ratio of 1∶1 (A) or 1∶100 (B). The competitive index was calculated as the ratio of mutant to wild-type recovered from the small intestine, corrected for the ratio of mutant to wild-type that was present in the inoculum. Each symbol represents one mouse. *The ΔRND mutant was not recovered from mice necessitating the calculation of a theoretical CI as described in the Materials and Methods. Mean and standard deviation are indicated by horizontal bars. Significance was determined using the Mann-Whitney U t-test. (1) p<0.01.

Figure 3. Colonization of the infant mouse small intestine by V. cholerae RND efflux mutants.

Infant mice were challenged with ∼6×10⁶ cfu (A) or ∼8×10⁷ cfu (B) of the indicated V. cholerae mutant. Bacterial loads in the small intestine were assessed after overnight incubation. Means and standard deviation are indicated by horizontal bars. The Mann Whitney U t-test was used to determine significance. (1) p<0.05 compared to all tested strains; (2) p<0.05 compared to ΔvexBDK; (3) p<0.05 compared to the ∼6×10⁶ cfu (A) ΔvexBDH challenge.