Disulfide Bond Formation and ToxR Activity in Vibrio cholerae

Vera H I Fengler,Sandro Roier,Joachim Reidl,Stefan Schild,Sarah Tutz,Hanna Ebner,Andrea Seper,Eva C Boritsch,Michael Hensel

doi:10.1371/journal.pone.0047756

Abstract

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.

Highlights

Vibrio cholerae is a Gram-negative, facultative anaerobic bacterium
To address whether ToxR activity depends on disulfide bond formation via the Dsb system [55], porin production was monitored by comparing a V. cholerae WT strain and corresponding dsbA, dsbB and dsbC deletion mutants
In vitro analysis using the purified periplasmic domain of ToxR showed that ToxR homodimers exist and rely on cysteine 293 by forming an intermolecular disulfide bond [59]. We revisited these earlier characterizations with our work, because we observed that dsbAB mutants affect porin production of OmpU and OmpT

Summary

Introduction

Vibrio cholerae is a Gram-negative, facultative anaerobic bacterium. It is the causative agent of cholera, which is endemic in India, Bangladesh, Southeast Asia, Africa and South America [1]. V. cholerae bacteria pass through the gastric acid compartment of the stomach, penetrate the mucus lining of the intestinal epithelia and start colonizing the small intestine. This compartment contains growth inhibitory substances, such as bile salts and organic acids and factors of the innate immune system, e.g., complement secreted by intestinal epithelial cells [4] and defensins produced by Paneth cells [5]. V. cholerae has developed the ability to survive, colonize and produce virulence factors [6] in spite of harsh stress conditions [7,8]

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Conclusion