Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
In: PLoS Pathogens, Jg. 4 (2008-01-18), Heft 1, p e4, S. e4
Online
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Zugriff:
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.
Author Summary Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic enteric pathogen that attaches to host cells and forms actin-filled membranous protrusions called pedestals. Pedestal formation is initiated when the EPEC virulence factor, Tir, is translocated into the host cell via bacterial Type III secretion (T3S) and inserted into the plasma membrane, initiating a signaling cascade that results in actin polymerization beneath attached bacteria. We have developed a cytoplasmic extract-based system in permeabilized cells to study these early events in EPEC pathogenesis, many of which are not easily studied in intact cells. We have taken advantage of the observation that EPEC fail to form pedestals on red blood cells (RBC). We report that low calcium triggers T3S of Tir into RBC, indicating how the T3S apparatus may sense entry into the host cytoplasm. Additionally, insertion of Tir into the host membrane depends upon host cytoplasmic components, a requirement not previously recognized or accessible to experimental manipulation in intact cells. Finally, cytoplasmic extract reconstitutes actin polymerization beneath attached bacteria using signaling molecules required in intact cells. We are currently purifying the components that mediate these processes. Together, these experiments show how functional biochemistry approaches can reveal novel roles for cytoplasmic factors in host–pathogen interactions.
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Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells
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Autor/in / Beteiligte Person: | Kalman, Daniel ; Swimm, Alyson |
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Zeitschrift: | PLoS Pathogens, Jg. 4 (2008-01-18), Heft 1, p e4, S. e4 |
Veröffentlichung: | Public Library of Science (PLoS), 2008 |
Medientyp: | unknown |
ISSN: | 1553-7374 (print) |
DOI: | 10.1371/journal.ppat.0040004 |
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