Diarrheal disease is a major health problem especially in developing countries where pathogenic Escherichia coli infections can cause over 1.5 million deaths annually. A common feature that increases the pathogenicity of important diarrheal pathogens such as enteropathogenic E. coli (EPEC) is the secretion of high-molecular-weight toxins known as serine protease autotransporters of Enterobacteriacea (SPATES). EspC is one of these SPATEs which enters enterocytes to cause tissue destruction. However, the molecular details of how EspC recognizes and enters epithelial cells are unknown. This present work investigates the molecular detail of EspC toxicity and how this protein recognizes and enters epithelial cells. This work will reveal mode of action of EspC in bacterial pathogenesis and finally define avenues to combat diarrheal diseases.
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 subdomains thought to be responsible for epithelial cell binding and internalization. Using deletion mutants of these protruding subdomains we uncovered that bacterial type III secretion system mediated internalization of EspC is completely independent of these subdomains. Unexpectantly, we also found that these loops are required for the protease activity and overall stability of this toxin.
Detailed understanding of the structural-function relationships of this toxin informs the design of inhibitors that block either EspC protease activity or enterocyte entry. To design anti-microbials that block the protease active site using small molecules, we have initially determined the EspC structure in complex with inhibitor PMSF and confirmed its in vitro inactivation. Further we have developed four nanobodies that specifically inhibit EspC protease activity and toxicity. One of them (NbH5) was found to have a dual action where it can block EspC internalization as well. We crystalize this NbH5 nanobody in complex with EspC and currently the structure determination is in progress.
Collectively this work is revealing the all-important molecular mechanisms of SPATEs, an important group of bacterial cytotoxins, and laying a foundation towards the structure-based discovery of inhibitors that combat diarrheal disease.