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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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Related in: MedlinePlus

Actin Colocalizes with Tir and Phosphotyrosine (PY) Staining in RBC Infected with EPEC and Exposed to Extract(A) Images of RBC infected with EPEC and exposed to either buffer or 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. Note that tyrosine phosphorylation and actin polymerization in extract-treated RBC is colocalized with Tir.(B) Quantitative analysis of colocalization of Tir and PY staining beneath attached EPEC in RBC exposed to either buffer or extract.(C) Quantitative analysis of colocalization of Tir, PY, and actin staining beneath attached EPEC in RBC exposed to either buffer or extract. Scale bars in all images represent 5 μm.
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ppat-0040004-g002: Actin Colocalizes with Tir and Phosphotyrosine (PY) Staining in RBC Infected with EPEC and Exposed to Extract(A) Images of RBC infected with EPEC and exposed to either buffer or 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. Note that tyrosine phosphorylation and actin polymerization in extract-treated RBC is colocalized with Tir.(B) Quantitative analysis of colocalization of Tir and PY staining beneath attached EPEC in RBC exposed to either buffer or extract.(C) Quantitative analysis of colocalization of Tir, PY, and actin staining beneath attached EPEC in RBC exposed to either buffer or extract. Scale bars in all images represent 5 μm.

Mentions: We next set out to determine whether intense actin staining seen beneath EPEC in RBC exposed to extract was characteristic of pedestals formed by EPEC on other cell types. As in HeLa or 3T3 cells infected with EPEC, Tir and phosphotyrosine colocalized with intense actin staining directly beneath attached bacteria on RBC treated with extract (Figure 2A). Quantitation of colocalization of Tir with phosphotyrosine and actin staining (see Materials and Methods) indicated that of those bacteria attached to RBC and expressing Tir, ∼60% were colocalized with phosphotyrosine (Figure 2B), and ∼45% with phosphotyrosine and intense actin staining (Figure 2C) following exposure to extract. Accordingly, the intense actin staining was only observed in conjunction with staining for bacteria, phosphotyrosine, and Tir. No colocalization of Tir with actin or phosphotyrosine was evident in RBC treated with buffer alone (Figure 2A). Thus, as in pedestals on intact cells, actin polymerization on extract treated RBC was colocalized with phosphotyrosine staining.


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

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

Actin Colocalizes with Tir and Phosphotyrosine (PY) Staining in RBC Infected with EPEC and Exposed to Extract(A) Images of RBC infected with EPEC and exposed to either buffer or 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. Note that tyrosine phosphorylation and actin polymerization in extract-treated RBC is colocalized with Tir.(B) Quantitative analysis of colocalization of Tir and PY staining beneath attached EPEC in RBC exposed to either buffer or extract.(C) Quantitative analysis of colocalization of Tir, PY, and actin staining beneath attached EPEC in RBC exposed to either buffer or extract. 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-g002: Actin Colocalizes with Tir and Phosphotyrosine (PY) Staining in RBC Infected with EPEC and Exposed to Extract(A) Images of RBC infected with EPEC and exposed to either buffer or 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. Note that tyrosine phosphorylation and actin polymerization in extract-treated RBC is colocalized with Tir.(B) Quantitative analysis of colocalization of Tir and PY staining beneath attached EPEC in RBC exposed to either buffer or extract.(C) Quantitative analysis of colocalization of Tir, PY, and actin staining beneath attached EPEC in RBC exposed to either buffer or extract. Scale bars in all images represent 5 μm.
Mentions: We next set out to determine whether intense actin staining seen beneath EPEC in RBC exposed to extract was characteristic of pedestals formed by EPEC on other cell types. As in HeLa or 3T3 cells infected with EPEC, Tir and phosphotyrosine colocalized with intense actin staining directly beneath attached bacteria on RBC treated with extract (Figure 2A). Quantitation of colocalization of Tir with phosphotyrosine and actin staining (see Materials and Methods) indicated that of those bacteria attached to RBC and expressing Tir, ∼60% were colocalized with phosphotyrosine (Figure 2B), and ∼45% with phosphotyrosine and intense actin staining (Figure 2C) following exposure to extract. Accordingly, the intense actin staining was only observed in conjunction with staining for bacteria, phosphotyrosine, and Tir. No colocalization of Tir with actin or phosphotyrosine was evident in RBC treated with buffer alone (Figure 2A). Thus, as in pedestals on intact cells, actin polymerization on extract treated RBC was colocalized with phosphotyrosine staining.

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