Introduction:
Diarrheal disease is a major health problem especially in developing countries where pathogenic Escherichia colicause 1.5 million deaths annually. Secretion of high-molecular-weight toxins known as serine protease autotransporters of Enterobacteriacea(SPATES) is a common virulence trait of enteropathogenic E. coli(EPEC) and enteroaggregative E. coli(EAEC), two major causative agents of diarrheal disease. EspC and Pet are two of these SPATEs which bind and enter epithelial cells to cause tissue destruction during infection. However, the molecular details of how these SPATEs recognize and enter epithelial cells are unknown.
Hypothesis & Aim:
To determine the structures and define the mechanisms of action of the EspC and Pet toxins for the development of antimicrobials against diarrheal disease.
Methodology:
A cross-disciplinary approach by combining cutting edge techniques in structural biology, microbiology and molecular biology.
Results & Conclusions:
We determined the crystal structure of EspC which was found to encompass a large 3-stranded b-helix with an N-terminal chymotrypsin-like serine protease domain. The large b-helix contains a number of loops thought to be responsible for epithelial cell binding and internalisation. Using the EspC structure we have designed deletion mutants of these protruding loops to uncover how this toxin hijacks the bacterial type III secretion system to specifically enter intestinal enterocytes. Unexpectantly, we found that these loops are also required for the protease activity & overall stability of these toxins. Detailed understanding of the structural-function relationships of this toxin will inform the design of inhibitors to block EspC from entering host cells and cause tissue destruction. The EspC structure is also a means to design anti-microbials that block the protease active site using small molecules. To this end we have also determined the EspC & Pet structures in complex with inhibitor PMSF and confirmed their in vitro inactivation. Collectively this work is revealing all important molecular details of the SPATEs mechanism of action and laying a foundation towards the structure-based drug discovery to combat diarrheal disease.