Eps15, but not AP‐2, is recruited and essential for the formation of enteropathogenic E. coli pedestals

Abstract

Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that cause serious infantile diarrhea. The hallmark of EPEC pathogenesis is its ability to colonize the surface of gut epithelial cells and inject effector proteins to hijack sub‐cellular components, forming characteristic actin‐rich pedestals beneath the attached bacteria. Efficient formation of pedestals requires the phosphorylation of the EPEC effector protein Tir at Tyrosine 474 (Y474). Clathrin, a key component in receptor‐mediated endocytosis, was previously found to participate in pedestal formation. We examined the clathrin‐associated endocytic proteins AP‐2 and its binding partner, Eps15. We found that Eps15 is recruited to the pedestal together with clathrin while AP‐2 is not. Further analysis using GFP‐Eps15 mutants revealed that such recruitment does not require the AP‐2 binding domain at the Eps15 C‐terminus, confirming previous results that pedestal forms efficiently in the absence of AP‐2. RNA interference showed that pedestal formation is inhibited in the absence of Eps15, suggesting Eps15 is required to generate pedestals. Finally we showed that Eps15 recruitment to pedestals required Tir Y474 phosphorylation. These results reveal a novel internalization subversion strategy employed by extracellular pathogens, supporting the recent paradigm that endocytic proteins are important for EPEC‐mediated disease.Grant Funding Source: CIHR