Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation

Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.The extracellular bacterial pathogen enteropathogenic Escherichia coli (EPEC) causes serious diarrheal disease in humans and is a prevalent microbe involved in childhood mortality in the developing world. This microbe is part of a larger family of bacteria called the attaching and effacing (A/E) pathogens that also includes the human-specific pathogen enterohemorrhagic E. coli (EHEC) and the murine disease-causing bacterium Citrobacter rodentium. These bacteria attach to intestinal epithelial cells and use a type III secretion system to directly deliver effector proteins from the bacterial cytosol into the cytoplasm of host cells. Among other functions, these effectors harness the host cell''s cytoskeleton (4) to generate actin-rich pedestals that are hallmarks of virulence for this class of pathogens (14). One of these effectors, the translocated intimin receptor (Tir), is key to pedestal formation. Following translocation into the host cell, Tir becomes embedded in the host cell plasma membrane, where its extracellular domain acts to firmly anchor the pathogen to the epithelial cell. In cultured cells, the intracellular cytoplasmic domain of EPEC Tir can become phosphorylated at tyrosine 474 (Y474) (6), where it recruits the adaptor protein Nck (7). These events all occur prior to actin filament polymerization beneath the attached bacteria via an N-WASP- and Arp2/3-based mechanism (7, 11). Although this is the prominent strategy used by EPEC to recruit actin to pedestals, a Y474-independent strategy also exists, but it occurs at a much lower frequency. During such instances, EPEC Tir becomes phosphorylated at Y454 and actin recruitment is independent of Nck (1).Previous work highlighted a role for clathrin during some bacterial infections (19, 20). Although the role of clathrin during enteropathogenic E. coli infections was not investigated until recently (20), the finding of clathrin at the tips of EPEC pedestals, coupled with the discovery of dynamin-2, another protein known to be involved in endocytosis, associated within the actin stalk of EPEC pedestals (18), suggests a possible role for additional endocytosis-associated proteins and indicates that a unique mechanism is employed by EPEC to remain extracellular despite the presence of these endocytic components. Other proteins, including the actin-associated protein cortactin, are also prominent at these structures. Cortactin is found throughout EPEC pedestals as well as pedestals formed by other attaching and effacing pathogens (2, 3). Thus, in order to further examine other endocytosis-associated proteins during EPEC pedestal formation, we opted to immunolocalize the endocytosis-related protein CD2AP (CD-2-associated protein) during these infections. CD2AP is a clathrin-associated endocytosis protein that directly associates with cortactin in other systems (12, 17).We discovered that CD2AP is present at EPEC pedestals and is a crucial component for their formation. Through the use of various host cell modification strategies, we subsequently explored the recruitment of the endocytosis-associated proteins at EPEC pedestals and found that during Nck-dependent pedestal formation, EPEC sequentially recruits Nck, clathrin, cortactin, and then CD2AP at the pedestal tip prior to the actin filament polymerization machinery at these sites.