| Enteropathogenic Escherichia coli (EPEC) is a gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhea. EPEC subverts the host cell actin cytoskeleton, causing a rearrangement of cytoskeletal components into a characteristic structure, the attaching and effacing lesion, or pedestal. EPEC transmits signals through the host cell plasma membrane via direct injection of virulence factors by the type III secretion system. One injected factor is Tir, which functions as the receptor for the EPEC outer membrane protein, intimin. Upon binding intimin, Tir initiates pedestal formation. Both the amino and carboxyl termini of Tir are oriented inside the host cell cytosol, where each mediates effects on the actin cytoskeleton. Tyrosine phosphorylation at the C-terminus of Tir is required for actin polymerization to occur.; A major goal of this thesis was to define the composition of the EPEC pedestal and determine which cytoskeletal components contributed to its formation. To this end, over 30 cytoskeletal proteins were identified in the pedestal tip and length. Recruitment of these proteins depended on either initial EPEC attachment, Tir insertion in the membrane, or Tir tyrosine phosphorylation.; Among the proteins recruited to EPEC pedestals were N-WASP and the Arp2/3 complex, key regulators of actin polymerization. N-WASP was recruited to the pedestal tip through its GTPase binding domain and initiated pedestals through its acidic domain by recruiting the Arp2/3 complex. These data suggest that N-WASP and the Arp2/3 complex are critical effectors of actin polymerization leading to pedestal formation.; To identify a binding partner of Tir, the yeast two-hybrid system and a Tir-affinity column were employed, resulting in the identification of α-actinin as a binding partner of Tir. Direct binding was confirmed by ELISA, far western, and co-immunoprecipitation. α-actinin overexpression in HeLa cells resulted in a two-fold increase in pedestal length suggesting it plays a key role in pedestal formation.; Collectively, these results demonstrate that the EPEC pedestal is a complex structure, with Tir playing the central role in mediating its formation. This work has significantly increased our understanding of the cytoskeletal components involved in pedestal formation, and provides insight into the mechanisms of disease for this important pathogen. |
