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

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 kin...

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Autores principales: Swimm, Alyson I, Kalman, Daniel
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211550/
https://www.ncbi.nlm.nih.gov/pubmed/18208322
http://dx.doi.org/10.1371/journal.ppat.0040004
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author Swimm, Alyson I
Kalman, Daniel
author_facet Swimm, Alyson I
Kalman, Daniel
author_sort Swimm, Alyson I
collection PubMed
description 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.
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spelling pubmed-22115502008-01-23 Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells Swimm, Alyson I Kalman, Daniel PLoS Pathog Research Article 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. Public Library of Science 2008-01 2008-01-18 /pmc/articles/PMC2211550/ /pubmed/18208322 http://dx.doi.org/10.1371/journal.ppat.0040004 Text en © 2008 Swimm and Kalman. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Swimm, Alyson I
Kalman, Daniel
Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
title Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
title_full Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
title_fullStr Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
title_full_unstemmed Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
title_short Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
title_sort cytosolic extract induces tir translocation and pedestals in epec-infected red blood cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211550/
https://www.ncbi.nlm.nih.gov/pubmed/18208322
http://dx.doi.org/10.1371/journal.ppat.0040004
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AT kalmandaniel cytosolicextractinducestirtranslocationandpedestalsinepecinfectedredbloodcells