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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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Exposure of RBC to Cytosolic Extract Induces Actin Polymerization beneath Attached EPEC(A) DIC image of EPEC (a) adhering to RBC that had undergone hemolysis (b) or remained intact (c) 6 h after infection.(B) Images of RBC infected with EPEC and stained with DAPI to visualize bacteria (blue), Alexa-488-phalloidin to visualize actin (green), anti-Tir antibody (left panel), and anti-phosphotyrosine (PY) 4G10 antibody (middle panel). Note the lack of PY and actin accumulation beneath the bacteria.(C) Schematic of method used to induce pedestal formation in RBC.(D) Images of EPEC infected RBC after exposure to either buffer or extract for 20 min. Inset in D depicts actin pedestals formed on a HeLa cell infected with EPEC. Cells are stained with DAPI to identify EPEC (blue) and Alexa-488 phalloidin to visualize actin (green). Scale bar in all images represents 5 μm.
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ppat-0040004-g001: Exposure of RBC to Cytosolic Extract Induces Actin Polymerization beneath Attached EPEC(A) DIC image of EPEC (a) adhering to RBC that had undergone hemolysis (b) or remained intact (c) 6 h after infection.(B) Images of RBC infected with EPEC and stained with DAPI to visualize bacteria (blue), Alexa-488-phalloidin to visualize actin (green), anti-Tir antibody (left panel), and anti-phosphotyrosine (PY) 4G10 antibody (middle panel). Note the lack of PY and actin accumulation beneath the bacteria.(C) Schematic of method used to induce pedestal formation in RBC.(D) Images of EPEC infected RBC after exposure to either buffer or extract for 20 min. Inset in D depicts actin pedestals formed on a HeLa cell infected with EPEC. Cells are stained with DAPI to identify EPEC (blue) and Alexa-488 phalloidin to visualize actin (green). Scale bar in all images represents 5 μm.

Mentions: In initial experiments, human RBC were plated and infected with EPEC from an overnight culture, as previously described [16,19]. In accordance with previous reports [16,17], the majority of RBC underwent hemolysis during infection and EPEC remained adhered to the lysed RBC membranes (Figure 1A). Upon visualization of Tir, phosphotyrosine and actin by indirect immunofluorescence microscopy, Tir was present beneath a small fraction of attached EPEC, but no associated phosphotyrosine or actin staining was evident, in agreement with previous reports [19] (Figure 1B), even with prolonged infections (up to 16 h, the longest time tested). By contrast, infection of HeLa cells with EPEC for as little as four hours induced formation of characteristic actin “pedestals”, visualized as intense actin staining apposed to the bacteria (Figure 1D; inset).


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

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

Exposure of RBC to Cytosolic Extract Induces Actin Polymerization beneath Attached EPEC(A) DIC image of EPEC (a) adhering to RBC that had undergone hemolysis (b) or remained intact (c) 6 h after infection.(B) Images of RBC infected with EPEC and stained with DAPI to visualize bacteria (blue), Alexa-488-phalloidin to visualize actin (green), anti-Tir antibody (left panel), and anti-phosphotyrosine (PY) 4G10 antibody (middle panel). Note the lack of PY and actin accumulation beneath the bacteria.(C) Schematic of method used to induce pedestal formation in RBC.(D) Images of EPEC infected RBC after exposure to either buffer or extract for 20 min. Inset in D depicts actin pedestals formed on a HeLa cell infected with EPEC. Cells are stained with DAPI to identify EPEC (blue) and Alexa-488 phalloidin to visualize actin (green). Scale bar in all images represents 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040004-g001: Exposure of RBC to Cytosolic Extract Induces Actin Polymerization beneath Attached EPEC(A) DIC image of EPEC (a) adhering to RBC that had undergone hemolysis (b) or remained intact (c) 6 h after infection.(B) Images of RBC infected with EPEC and stained with DAPI to visualize bacteria (blue), Alexa-488-phalloidin to visualize actin (green), anti-Tir antibody (left panel), and anti-phosphotyrosine (PY) 4G10 antibody (middle panel). Note the lack of PY and actin accumulation beneath the bacteria.(C) Schematic of method used to induce pedestal formation in RBC.(D) Images of EPEC infected RBC after exposure to either buffer or extract for 20 min. Inset in D depicts actin pedestals formed on a HeLa cell infected with EPEC. Cells are stained with DAPI to identify EPEC (blue) and Alexa-488 phalloidin to visualize actin (green). Scale bar in all images represents 5 μm.
Mentions: In initial experiments, human RBC were plated and infected with EPEC from an overnight culture, as previously described [16,19]. In accordance with previous reports [16,17], the majority of RBC underwent hemolysis during infection and EPEC remained adhered to the lysed RBC membranes (Figure 1A). Upon visualization of Tir, phosphotyrosine and actin by indirect immunofluorescence microscopy, Tir was present beneath a small fraction of attached EPEC, but no associated phosphotyrosine or actin staining was evident, in agreement with previous reports [19] (Figure 1B), even with prolonged infections (up to 16 h, the longest time tested). By contrast, infection of HeLa cells with EPEC for as little as four hours induced formation of characteristic actin “pedestals”, visualized as intense actin staining apposed to the bacteria (Figure 1D; inset).

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