The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides

Abstract

BPI (bactericidal/permeability-increasing) is a potent antimicrobial protein that was recently reported to be expressed as a surface protein on human gastrointestinal tract epithelial cells. In this study, we investigated the resistance of Vibrio cholerae, a small-bowel pathogen that causes cholera, to a BPI-derived peptide, P2. Unlike in Escherichia coli and Salmonella enterica serovar Typhimurium, resistance to P2 in V. cholerae was not dependent on the BipA GTPase. Instead, we found that ToxR, the master regulator of V. cholerae pathogenicity, controlled resistance to P2 by regulating the production of the outer membrane protein OmpU. Both toxR and ompU mutants were at least 100-fold more sensitive to P2 than were wild-type cells. OmpU also conferred resistance to polymyxin B sulfate, suggesting that this porin may impart resistance to cationic antibacterial proteins via a common mechanism. Studies of stationary-phase cells revealed that the ToxR-repressed porin OmpT may also contribute to P2 resistance. Finally, although the mechanism of porin-mediated resistance to antimicrobial peptides remains elusive, our data suggest that the BPI peptide sensitivity of OmpU-deficient V. cholerae is not attributable to a generally defective outer membrane.

FIG. 1.

Dose-dependent killing of V. cholerae by the BPI peptide P2. Mid-log-phase V. cholerae (O395 ΔlacZ) and stationary-phase V. cholerae O395 lacZ⁺ cells were incubated with the indicated concentrations of P2 for 90 min. The numbers of CFU of the cells were determined by plating on LB agar containing X-Gal, and the percent survival of the cells was calculated by normalizing to the control (0 P2) at each time point. The means of three independent experiments and corresponding standard errors of the means (SEMs) are presented.

FIG. 2.

ToxR, but not ToxT, contributes to resistance to BPI peptide in V. cholerae. The percent survival of mid-log-phase wt, toxR, and toxT V. cholerae cells as a function of time of incubation with 40 mg of P2 per ml is shown. Shown here are the means of three experiments with SEMs.

FIG. 3.

OmpU confers BPI peptide resistance in mid-log-phase cells. (A) The percent survival of mid-log-phase wt O395 and ompU, ompT, ompU/ompT, and ompU+pU−ompU cells as a function of time of incubation with 40 μg of P2 per ml is shown. Means of three independent experiments and corresponding SEMs are presented. (B) Western blots comparing OmpU and OmpT levels in stationary- and mid-log-phase cultures of the indicated strains. The asterisk denotes a cross-reacting band used as a loading control.

FIG. 4.

Ectopic expression of OmpU as well as OmpT imparts resistance to BPI peptide in stationary-phase O395 cells. (A) The percent survival of stationary-phase wt O395 (⧫) and isogenic toxR (▪), ompT (▴), ompU+pU−ompT (×), toxR + pT−ompT (✻), and ompU (•) cells is shown as a function of time of incubation with 40 μg of P2 per ml. The means of three independent experiments and corresponding SEMs are presented. (B) Western blots showing OmpT and OmpU levels in these strains at stationary phase. The asterisk denotes a cross-reacting band used as a loading control. Y= denotes another cross-reactive band just above the OmpT band.

FIG. 5.

OmpU also confers resistance to polymyxin B sulfate. The percent survival of mid-log-phase O395 wt, ompU, and ompU+pU−ompU cells is shown as a function of time of incubation with 12 μg of polymyxin B sulfate per ml. Shown here are means of two independent experiments.

FIG. 6.

BPI peptide increases OM permeability in wt and ompU O395 to similar extents. Nitrocefin hydrolysis was monitored at an OD₄₉₀ as a function of time and used as a measure of OM permeability. In the absence of P2, there was minimal nitrocefin hydrolysis in wt and ompU cells. Data are representative of five independent experiments.