Abstract
A type VI secretion system (T6SS) was recently shown to be required for full virulence of Vibrio cholerae O37 serogroup strain V52. In this study, we systematically mutagenized each individual gene in T6SS locus and characterized their functions based on expression and secretion of the hemolysin co-regulated protein (Hcp), virulence towards amoebae of Dictyostelium discoideum and killing of Escherichia coli bacterial cells. We group the 17 proteins characterized in the T6SS locus into four categories: twelve (VipA, VipB, VCA0109–VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two (VasH and VCA0122) are regulators that are required for T6SS gene expression and virulence; another two, VCA0121 and valine-glycine repeat protein G 3 (VgrG-3), are not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; the last group is represented by VCA0118, which is not required for Hcp expression or secretion but still plays a role in both amoebae and bacterial killing and may therefore be an effector protein. We also showed that the clpV gene product is required for Dictyostelium virulence but is less important for killing E. coli. In addition, one vgrG gene (vgrG-2) outside of the T6SS gene cluster was required for bacterial killing but another (vgrG-1) was not. However, a bacterial killing defect was observed when vgrG-1 and vgrG-3 were both deleted. Several genes encoded in the same putative operon as vgrG-1 and vgrG-2 also contribute to virulence toward Dictyostelium but have a smaller effect on bacterial killing. Our results provide new insights into the functional requirements of V. cholerae's T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.
Highlights
Pathogenicity of bacteria is often critically dependent on secretion systems to export toxic molecules into the environment or translocate effectors into the host cells
We examined whether VCA0118 was required for the secretion of valine-glycine repeat protein G (VgrG)-1, the only T6SS effector required for Dictyostelium and macrophage virulence so far reported for V. cholerae [20]
We characterized each mutant for hemolysin co-regulated protein (Hcp) expression and secretion, and examined their contributions to virulence with Dictyostelium amoebae and E. coli
Summary
Pathogenicity of bacteria is often critically dependent on secretion systems to export toxic molecules into the environment or translocate effectors into the host cells. At least six different secretion systems (type I through type VI) have been found in Gram-negative bacterial pathogens of animals and plants [1,2,3] These secretion systems are distinguished in part by the conserved structural components that define them and by the characteristics of their substrates and the subcellular paths that their substrates take during the export process. In most T6SSs, hemolysin co-regulated protein (Hcp) and valine-glycine repeat protein G (VgrG) are exported by T6SS They are proposed to be the extracellular components of T6SS apparatus, and their secretion is codependent [6,10,20,21,22]. In E. tarda, a systematic mutation of one T6SS cluster has been performed and three categories of proteins have been identified, including 12 genes required for the Hcp secretion [10]
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