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

Swimm AI, Kalman D - PLoS Pathog. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, United States of America.

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.

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Actin Polymerization Induced by Extract in EPEC-Infected RBC Utilizes Both Bacterial and Host Factors(A) Images of RBC infected with EPEC mutants lacking Tir (EPECΔtir) or intimin (EPECΔeae) and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488 phalloidin to visualize actin, and either anti-Tir antibody or anti-PY 4G10 antibody.(B) Images of RBC infected with EPEC and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488-phalloidin to visualize actin, and either anti–Nck antibody, anti-WASP antibody, or anti-Arp 2/3 p21 antibody. Scale bars in all images represent 5 μm.
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ppat-0040004-g003: Actin Polymerization Induced by Extract in EPEC-Infected RBC Utilizes Both Bacterial and Host Factors(A) Images of RBC infected with EPEC mutants lacking Tir (EPECΔtir) or intimin (EPECΔeae) and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488 phalloidin to visualize actin, and either anti-Tir antibody or anti-PY 4G10 antibody.(B) Images of RBC infected with EPEC and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488-phalloidin to visualize actin, and either anti–Nck antibody, anti-WASP antibody, or anti-Arp 2/3 p21 antibody. Scale bars in all images represent 5 μm.

Mentions: Interactions between intimin, located in the outer membrane of EPEC, and Tir, in the plasma membrane of the infected cell, induce Tir clustering, and are required for pedestal formation and for efficient phosphorylation of Tir [6]. To determine whether interactions between Tir and intimin were required for actin polymerization induced by extract in RBC, we tested EPEC strains deficient in either Tir (EPECΔtir) or intimin (EPECΔeae) for their ability to induce actin polymerization after treatment with extract. As seen in Figure 3A, both EPECΔeae and EPECΔtir failed to induce tyrosine phosphorylation or actin polymerization apposed to bacteria after exposure to extract. Together these data suggest that, as in other models of EPEC infection, interactions between Tir and intimin are critical for actin polymerization induced beneath EPEC in RBC exposed to extract.


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

Swimm AI, Kalman D - PLoS Pathog. (2008)

Actin Polymerization Induced by Extract in EPEC-Infected RBC Utilizes Both Bacterial and Host Factors(A) Images of RBC infected with EPEC mutants lacking Tir (EPECΔtir) or intimin (EPECΔeae) and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488 phalloidin to visualize actin, and either anti-Tir antibody or anti-PY 4G10 antibody.(B) Images of RBC infected with EPEC and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488-phalloidin to visualize actin, and either anti–Nck antibody, anti-WASP antibody, or anti-Arp 2/3 p21 antibody. Scale bars in all images represent 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211550&req=5

ppat-0040004-g003: Actin Polymerization Induced by Extract in EPEC-Infected RBC Utilizes Both Bacterial and Host Factors(A) Images of RBC infected with EPEC mutants lacking Tir (EPECΔtir) or intimin (EPECΔeae) and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488 phalloidin to visualize actin, and either anti-Tir antibody or anti-PY 4G10 antibody.(B) Images of RBC infected with EPEC and treated with extract. Cells are labeled with DAPI to identify EPEC, Alexa-488-phalloidin to visualize actin, and either anti–Nck antibody, anti-WASP antibody, or anti-Arp 2/3 p21 antibody. Scale bars in all images represent 5 μm.
Mentions: Interactions between intimin, located in the outer membrane of EPEC, and Tir, in the plasma membrane of the infected cell, induce Tir clustering, and are required for pedestal formation and for efficient phosphorylation of Tir [6]. To determine whether interactions between Tir and intimin were required for actin polymerization induced by extract in RBC, we tested EPEC strains deficient in either Tir (EPECΔtir) or intimin (EPECΔeae) for their ability to induce actin polymerization after treatment with extract. As seen in Figure 3A, both EPECΔeae and EPECΔtir failed to induce tyrosine phosphorylation or actin polymerization apposed to bacteria after exposure to extract. Together these data suggest that, as in other models of EPEC infection, interactions between Tir and intimin are critical for actin polymerization induced beneath EPEC in RBC exposed to extract.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, United States of America.

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.

Show MeSH
Related in: MedlinePlus