RNA-Based Regulation of the Type 3 Secretion Machine of Enterohaemorrhagic E. Coli

My lab has an ongoing interest in how complex genetic traits such as virulence are regulated and selected in bacterial pathogens. Non-coding RNA (ncRNA) regulation has come to the fore with the advent of RNA sequencing and it has been demonstrated that bacterial pathogens produce hundreds of ncRNAs. However, we have a poor understanding of the function of the majority of these RNA species. The functions of bacterial ncRNAs are likely to be exceptionally diverse, and we are using UV-crosslinking and deep sequencing techniques to study these processes and reveal novel mechanisms of gene regulation.

Enterohaemorrhagic E. coli (EHEC) causes sporadic outbreaks of severe diarrheal disease that may lead to renal failure and death. The major virulence factors responsible for human disease are the Shiga toxins (that cause kidney damage) and a type 3 secretion (T3S) system that allows the bacterium to attach to human cells. We have recently demonstrated that EHEC carries approximately 145 regulatory small RNAs and some appear to control expression of the T3S system. Using new CRISPRi technologies, this project will construct a library of small RNA knockdowns in EHEC and determine which small RNAs control expression of the T3S system.

The ability of small RNA knockdowns to colonize human cells will be assayed using tissue culture adhesion assays, and sRNA regulation confirmed using gene deletions, GFP translational fusions, and RNA immunoprecipitation techniques.

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