Molecular analysis of regulatory factors and environmental signals controlling secretion systems important for Yersinia virulence

Abstract

Type VI secretion systems are versatile nanomachines that exhibit structural similarity to bacteriophage tails. Bacteria use their T6SS to deliver toxic effectors into prokaryotic or eukaryotic cells. Most commonly, bacteria use their T6SS to compete with other bacteria in order to colonize a specific niche. Since assembly, contraction and disassembly of T6SSs is energetically costly for bacteria, expression of these systems is required to be under tight regulation. Recently, the T6SS4 of Y. pseudotuberculosis was linked to early virulence gene expression. In this context, the newly discovered regulator RovC was identified to be a novel activator of the T6SS4. The present study indicated that RovC is a crucial and direct activator of T6SS4 expression that interacts with the T6SS4 promoter. In addition, analysis of RovC proteins harboring different amino acid substitutions revealed that its DNA-binding region is located at the protein surface. Binding of RovC to the T6SS4 promoter is essential in order to induce expression of the operon. The present study could also demonstrate that the T6SS4 of Y. pseudotuberculosis is functional in vivo, since translocation of Hcp into the supernatant was detected in a RovC-dependent manner. Moreover, expression of the T6SS4 was reported to be controlled by the carbon storage regulator (Csr) system. The Csr system consists of the RNA-binding protein CsrA and the small nc RNAs, CsrB and CsrC. CsrA controls a large number of genes and is essential for Y. pseudotuberculosis to cause an infection in mice, and recently, it was implicated in the regulation of the T6SS4. The present work revealed that CsrA-dependent regulation of the T6SS4 is mediated via RovC. On the transcriptional level expression of rovC is indirectly repressed by CsrA. However, CsrA exerts a dual function since post-transcriptionally, CsrA is essential for rovC mRNA stabilization via direct interaction with the rovC transcript. In addition, regulation of the T6SS4 has been shown to be mediated by different regulators in Y. pseudotuberculosis. Among these are the response regulator OmpR, the virulence regulator RovM and the gene silencing protein H-NS. RovM was shown to strongly activate expression a T6SS4’-’lacZ reporter fusion comparable to RovC. However, this work revealed that the presence of RovM is not essential for the activation of the T6SS4, as deletion of rovM can be overcome by ectopic expression of RovC in a rovM mutant but not vice versa.