Cytosolic extract induces Tir translocation and pedestals in EPEC-infected red blood cells.

PLoS Pathogens
Alyson Swimm, Daniel Kalman

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 com...Continue Reading

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Citations

Apr 22, 2014·Microbial Pathogenesis·Fengyi Tang, Milton H Saier
Feb 26, 2010·Journal of Virology·Alyson I SwimmDaniel Kalman
Dec 17, 2008·Future Microbiology·Sigrid D AuweterB Brett Finlay
Dec 8, 2009·Nature Reviews. Microbiology·Matthew A Croxen, B Brett Finlay
Sep 27, 2019·Antibiotics·Heather A Pendergrass, Aaron E May

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Methods Mentioned

BETA
fluorescence microscopy
immunoprecipitation
dissection

Software Mentioned

Intelligent Imaging Innovations
ImageJ densitometry

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