Abstract:
Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections, causing a serious threat to global public health. The ability of UPEC strains to sense and adapt to host-associated environmental pressures, with the help of tightly regulated signalling systems, is critical for rapid colonisation and infection. Among these, the PhoQ/PhoP two-component system is essential for bacterial adaptation, regulating genes involved in membrane remodelling, stress resistance, and virulence. This Master’s thesis project focuses on signalling and pathogenicity in UPEC strains, specifically on investigating the direct regulatory network of the PhoQ/PhoP two-component system. In this study, we investigate potential targets for direct PhoP regulation using a combination of biochemical and genetic approaches, to better understand how the PhoQ/PhoP system regulates genes involved in virulence. Candidate genes for PhoPQ-dependent regulation were first identified by analysis of a proteomics dataset. To determine whether these genes are directly regulated by PhoP, Electrophoretic Mobility Shift Assays (EMSAs) were performed using purified and phosphorylated PhoP protein. These experiments demonstrated direct binding of PhoP to the promoters of selected target genes in vitro. Furthermore, to facilitate the genome-wide identification of PhoP-binding sites in vivo, a system with a FLAG-tagged PhoP was constructed and functionally validated for use in downstream chromatin immunoprecipitation (ChIP) experiments Together, these results establish an experimental framework to expand our current understanding of the PhoQ/PhoP regulon in uropathogenic E. coli and elucidate key regulatory elements of E. coli pathogenicity. This study also provides a basis to identify novel target sites for therapeutic strategies by uncovering additional players involved in bacterial virulence.
