Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells

• Type of Material: Article
• Authors: Daniel Kalman, Emory University; Alyson Swimm, Emory University
• Language: English
• Date: 2008-01
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2008 Swimm and Kalman.
• License: Creative Commons Attribution 3.0 Unported
• Final Published Version (URL): http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.0040004
• Title of Journal or Parent Work: PLoS Pathogens
• ISSN: 1553-7366
• Volume: 4
• Issue: 1
• Start Page: e4
• End Page: e4
• Grant/Funding Information: The work was supported by grant R01-AI056067–01 from the NIAID National Institute of Allergy and Infectious Diseases to DK.
• Research Categories: Biology, Microbiology; Health Sciences, Pathology

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.