Structural and Functional Insights into the Type II Secretion System of Vibrio cholerae.

Abstract

The bacterium Vibrio cholerae is the causative agent of cholera, a severe, acute diarrheal disease endemic throughout parts of the world. V. cholerae uses the type II secretion (T2S) system to transport the virulence factor cholera toxin to the extracellular milieu, which is primarily responsible for the disease’s hallmark massive, watery diarrhea. This widespread T2S system is structurally homologous to the type IV pilus (T4P) system. In this study, I use a suite of biochemical and genetic techniques to further elucidate the mechanism of the ATPase that powers T2S, EpsE, as well as the overall role of the T2S system in cell envelope stability.

EpsE contains a unique metal-binding (CM) domain that coordinates zinc via a tetracysteine motif. The CM domain is conserved among homologous T4P ATPases that power pilus assembly, but not T4P retraction ATPases. In order to assess the contribution of the CM domain to T2S, we removed the domain or substituted combinations of cysteine residues in the tetracysteine motif. All of these mutations abrogate EpsE’s ability to support T2S and have a dominant negative effect on secretion in the presence of WT EpsE. Additionally, EpsE’s ATPase activity is abolished upon zinc depletion in vitro. However, swapping the residues between the two dicysteine motifs with those from the homologue XcpR from Pseudomonas aeruginosa, resulting in the substitution of 17 out of 29 residues, has no significant effect on EpsE. Thus, while zinc coordination is essential for function, the CM domain may not play a species-specific role in EpsE and other T2S ATPases.

The eps genes encoding proteins required for T2S are putatively essential in V. cholerae, and eps inactivation results in widespread cell envelope defects, in addition to loss of secretion. To investigate the possibility that suppressor mutations facilitate eps gene inactivation, we used high-throughput genome sequencing to identify secondary mutations in V. cholerae eps mutants. Two independently constructed eps mutants contain distinct inactivating mutations in the T2-secreted protease VesC that may protect the cell from unwanted proteolysis by mislocalized VesC, suggesting one mechanism by which V. cholerae creates permissive conditions for acquiring eps mutations.