A novel function of enteropathogenic Escherichia coli secreted protein F within intestinal epithelial cells

Enteropathogenic E. coli (EPEC) are a leading cause of infantile diarrhea in developing countries, resulting in hundreds of thousands of deaths each year. EPEC infection requires the injection of effector proteins directly into host intestinal epithelial cells (IECs) via type three secretion systems. Secreted effectors interfere with IEC signaling pathways to induce several phenotypes, ultimately leading to disease.;Among the known EPEC effectors is E. coli secreted protein F (EspF), which is implicated in several phenotypes detrimental to the host, including disruption of epithelial barrier function. The mechanism by which this occurs was the focus of this investigation. An EspF mutant defective for mitochondrial targeting and apoptosis was functional in assays of epithelial barrier function, indicating that disruption of barrier function does not require EspF-induced apoptosis.;The possibility that EspF induces endocytosis of proteins required for maintaining cell-cell junctions, leading to loss of barrier function was examined. Pharmacological inhibition of endocytosis did not preserve barrier function nor was endocytosis of junctional proteins detected in response to EPEC infection, suggesting that EspF-dependent barrier disruption is independent of endocytosis.;EspF interacts with the endocytic regulator sorting nexin 9 (SNX9). This interaction alters SNX9 endocytic activity, inducing the formation of tubular plasma membrane invaginations. Thus, the second goal of this project was to characterize the mechanism of EspF-dependent SNX9 activation and to assess the significance of EspF/SNX9 interaction with regard to EPEC infection. EspF/SNX9 interaction was dispensable for intestinal barrier disruption, antiphagocytosis and NHE3 downregulation, however it was required for EPEC invasion of IECs. EspF/SNX9-dependent membrane tubulation was sensitive to inhibitors of clathrin coated pit (CCP) assembly and required functional lipid-binding domains of SNX9 as well as at least two SNX9 binding sites within EspF. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of CCP assembly. Inhibition of CCP assembly also blocked EPEC invasion, suggesting that EspF promotes EPEC invasion of IECs by harnessing the membrane-deforming activity of SNX9.