Regulation of the Yersinia pseudotuberculosis Type III Secretion System by the CpxAR Two-Component System

The type III secretion system (T3SS) is a cell envelope-spanning injectisome found in many Gram negative pathogens. Expression of the T3SS is controlled by extracellular signals such as cell envelope stress. For example, the CpxAR two component system negatively regulates the Yersinia pseudotuberculosis Ysc T3SS, although the mechanism for this regulation remains unclear. As expected from previous studies, we found that mutations in Y. pseudotuberculosis that lock CpxR in its constitutively activated state (?cpxA and cpxRD51E) led to decreased expression of the Ysc T3SS. CpxR did not bind to the regulatory regions of lcrF, encoding the Ysc T3SS master regulator. However, overexpression of LcrF protein rescued the T3SS expression defect of the ?cpxA and cpxRD51E mutants, suggesting that CpxR represses lcrF through an indirect mechanism. Transcriptome analysis of Y. pseudotuberculosis showed that 101 genes were upregulated and 77 genes downregulated in both the ?cpxA and cpxRD51E strains compared to wildtype, including seven genes known to modulate transcription. Individual deletion of these seven regulatory factors did not identify any single gene responsible for CpxR-dependent repression of LcrF, suggesting that the CpxAR system may act through multiple pathways to indirectly regulate the Ysc T3SS. However, this analysis led us to examine the role of the OmpR/EnvZ two component regulatory system, which is responsive to osmolarity, in LcrF regulation. Deletion of ompR led to a two-fold increase in LcrF and T3SS expression. These results suggest more complex regulation of the Ysc T3SS by two component regulatory systems than previously appreciated. Overall design: Yersinia pseudotuberculosis IP2666pIB1 strains with consitutive CpxR activation were generated, one lacking the cpxA gene (delta cpxA) and one posessing a cpxR allele coding for a phosphomemetic version of the protein (cpxRD51E). Mutant and wildtype strains were grown shaking at 26°C overnight in 2ml LB media and then subcultured to an OD of 0.2 in fresh low calcium LB supplemented with 20mM sodium oxalate and 20mM magnesium cholride. These cultures were grown shaking at 26°C for 1.5 hours and then shifted to 37°C shaking for 1.5 hours to mimic host infection conditions and induce Type III Secretion. Each sample was then pelleted and RNA extracted for sequencing.