Introduction:
Diarrhoeal diseases mostly attributable to pathogenic E. coli cause almost 1.5 million deaths annually, comprising 35% of all deaths due to bacterial infections. Both enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) are common diarrhoeal pathogens. A common feature that increases the pathogenicity of EPEC and EAEC is the secretion of high-molecular-weight toxins known as serine protease autotransporters of Enterobacteriacea (SPATES). EspC and Pet are two of these SPATEs which bind and enter epithelial cells to cause cytotoxic damage during infections. However, the molecular details of how these SPATEs recognize and enter epithelial cells is unknown.
Hypothesis & Aim:
To determine the structures of both the EspC and Pet toxins and define their mechanisms of action for the development of antimicrobials that target these toxins.
Methodology:
A cross-disciplinary approach by combining cutting edge techniques in structural biology, microbiology and molecular biology.
Results & Conclusions:
I have determined the first crystal structure of EspC and functionally characterized its serine protease activity. Importantly, this toxin displays some structural differences and activity profile to that of the other SPATEs. Using this information, I wish to understand how this toxin hijacks the bacterial type III secretion system to specifically enter intestinal enterocytes to cause tissue destruction and disease. Importantly, I am also working to understand how to inhibit the SPATE toxins in the development of antimicrobials. To this end I have also determined the EspC structure in complex with an inhibitor and confirmed in vitro its inactivation. This first structure of an inhibitor bound SPATE is revealing important molecular details for the structure based drug design of further inhibitors. In addition, I have also obtained diffracting crystals of the SPATE Pet in complex with bound inhibitor. Overall, my research is increasing the understanding of these important toxins and will lead to the development of antimicrobials to treat diarrhoeal diseases.