Like many Gram-negative bacteria, the food-borne pathogen Yersinia pseudotuberculosis harbours different regulatory mechanisms to maintain an intact bacterial envelope especially during exposure to extracytoplasmic stress (ECS). The CpxA-CpxR two component regulatory system is one such ECS-responsive regulatory mechanism. Activation of CpxA-CpxR two-component regulatory system (TCRS) accumulates phosphorylated CpxR (CpxR~P), which not only up-regulates various factors that are designed to maintain envelope integrity, but also down-regulates key determinants of bacterial virulence. Y. pseudotuberculosis establishes close host cell contact in part through the expression of the invasin adhesin. Invasin expression is positively regulated by the transcriptional regulator RovA, which in turn is negatively regulated in response to nutrient stress by a second transcriptional regulator RovM. In Y. pseudotuberculosis, loss of CpxA phosphatase activity accumulates CpxR~P, and this represses both rovA and inv transcription directly, or indirectly via activation of rovM transcription. It is now of interest to understand the molecular mechanism behind how CpxR~P regulates gene transcription both positively and negatively. A type III secretion system (T3SS) is a highly conserved multi-protein secretion system used by many Gram-negative bacteria to secrete protein cargo that counteracts the effects of a host cell emitted anti-bacterial activity. A typical set of proteins that make-up a functional T3SS includes structural proteins, translocators, effectors and regulatory proteins. Accumulation of CpxR~P was shown to repress the plasmid encoded Ysc-Yop T3SS of Y. pseudotuberculosis. Although yet to be confirmed experimentally, promoter-CpxR~P binding studies indicate multiple modes of regulatory control that for example, could influence levels of the plasmid-encoded Ysc-Yop system transcriptional activator, LcrF, and the chromosomal encoded negative regulators YmoA and YtxR.  Regulatory processes of TCRS involve transient molecular interactions between different proteins and also protein with DNA. Protein-protein interaction studies using the BACTH assay showed that it can be useful in analysing the molecular interactions involving the N-terminal domain of CpxR, while the λcI homodimerization assay can be useful in analysing molecular interactions involving the C-terminal domain of CpxR. Therefore, in combination with other biochemical and physiological tests, these hybrid-based assays can be useful in dissecting molecular contacts that can be helpful in exploring the mechanism behind CpxR~P mediated transcriptional regulation. In conclusion, this work uncovered direct involvement of CpxR~P in down-regulating virulence in Yersinia pseudotuberculosis. It also utilised genetic mutation and explored different protein-protein interaction assays to begin to investigate the mechanism behind the positive and negative regulation of gene expression mediated through active CpxR~P.

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