Abstract
Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food, and induce protrusion of actin-filled membranous pedestals beneath themselves upon attachment to intestinal epithelia. Pedestal formation requires clustering of Tir and subsequent recruitment of cellular tyrosine kinases including Abl, Arg, and Etk as well as signaling molecules Nck, N-WASP, and Arp2/3 complex. We have developed a cytosolic extract-based cellular system that recapitulates actin pedestal formation in permeabilized red blood cells (RBC) infected with EPEC. RBC support attachment of EPEC and translocation of virulence factors, but not pedestal formation. We show here that extract induces a rapid Ca++-dependent release of Tir from the EPEC Type III secretion system, and that cytoplasmic factor(s) present in the extract facilitate translocation of Tir into the RBC plasma membrane. We show that Abl and related kinases in the extract phosphorylate Tir and that actin polymerization can be reconstituted in infected RBC following addition of cytosolic extract. Reconstitution requires the bacterial virulence factors Tir and intimin, and phosphorylation of Tir on tyrosine residue 474 results in the recruitment of Nck, N-WASP, and Arp2/3 complex beneath attached bacteria at sites of actin polymerization. Together these data describe a biochemical system for dissection of host components that mediate Type III secretion and the mechanisms by which complexes of proteins are recruited to discrete sites within the plasma membrane to initiate localized actin polymerization and morphological changes.
Highlights
Pathogenic E. coli strains, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), contaminate food and water supplies and colonize the gastrointestinal tract, causing inflammation, diarrhea and death, among children in developing nations [1]
provides a means to reconstitute pedestal formation using purified components to help further characterize the dynamics of signaling events associated with
The EPEC strain E2348/ 69 was used for infections
Summary
Pathogenic E. coli strains, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), contaminate food and water supplies and colonize the gastrointestinal tract, causing inflammation, diarrhea and death, among children in developing nations [1]. Interaction with intimin clusters Tir, a process that is required for localized cytoskeletal changes [4,6,7], thereby initiating a signaling cascade involving recruitment of tyrosine kinases which phosphorylate Tir [4,8,9], and likely other bacterial or host proteins [7]. Stable kinase recruitment via polyproline and phosphotyrosine residues within Tir, and SH3 and SH2 domains within the kinases, is required for recruitment of downstream effectors and for pedestal formation [10] Such effectors include the host signaling molecules Nck [14], N-WASP, and the Arp2/3 complex [15], which catalyze actin polymerization beneath attached bacteria
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