Enteropathogenic Escherichia coli Secreted Effectors Perturb Endoplasmic Reticulum-Mitochondria Contact Points

Abstract

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative gut bacterial pathogen. EPEC is a leading cause of diarrheal mortality in children under 5 years of age. The main virulence determinant of EPEC is the ‘syringe-like’ type 3 secretion system (T3SS) through which the bacteria inject at least 23 ‘effector’ proteins directly into enterocytes to modulate host cell functions. These effectors modulate major host cell functions involving mitochondria, ER, junctions and cytoskeleton. Our time-lapse confocal imaging showed that cellular contact junctions between ER and mitochondria are uncoupled in late stages of EPEC infections. Furthermore, proteomics and western blot studies by our laboratory implicated a role for late T3SS effector EspH in upregulation of mitochondrial fission protein FIS1 which is a part of ER-mitochondria contact site in host cells. EPEC-EspH contributes to EPEC virulence in animal models of infection. FIS1, causes mitochondrial fission in host cells and regulates ER functions via its interaction with ER protein BAP31. Western blots in our lab also showed that EspH induces a truncated BAP31 form in infected host cells. FIS1-BAP31 complex is involved in calcium homeostasis between mitochondria and ER. As EspH disrupts this complex, we hypothesized that calcium homeostasis is disrupted in EPEC-infected cells. Our time lapse calcium imaging data show that there is a robust calcium release into the cytosol in later stages of the infections with a marginal decrease in ER calcium levels. ER calcium stores are required for its protein folding functions. Calcium depletion, therefore, leads to proteostasis which results in ER stress. Host cells activate unfolded protein response (UPR) to counter ER stress. However, inhibition or prolonging of UPR can cause the cell to switch from survival to apoptosis. Using western blots, we showed that EspH prevents activation of UPR proteins, IRE1 and PERK but selectively activates ATF6. Additionally, propidium-iodide uptake studies show that EspH promotes host cell death via BAP31. In conclusion, our data suggests that EspH likely causes host cell death via BAP31 cleavage-induced dysregulated ER stress response. This study provides insight into the role of ER-mitochondria contact points in extracellular bacteria pathogenesis.