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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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A Heat-Labile Cytosolic Factor(s) and Intimin Facilitate Translocation of Tir into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, extract, or heat-treated extract that was incubated at (65 °C for 15 min (Extract 65°) or incubated at 90 °C for 5 min (Extract 90°)), or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining after exposure to either DMEM, buffer, extract, or heat-treated extract for 20 min.(C) Western analysis of RBC infected with EPEC and exposed to either buffer, extract, heat-treated extract, heat-treated extract plus 5 mM CaCl2, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of RBC infected with EPEC or EPECΔeae and treated with either DMEM or extract for 20 min, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.
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ppat-0040004-g008: A Heat-Labile Cytosolic Factor(s) and Intimin Facilitate Translocation of Tir into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, extract, or heat-treated extract that was incubated at (65 °C for 15 min (Extract 65°) or incubated at 90 °C for 5 min (Extract 90°)), or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining after exposure to either DMEM, buffer, extract, or heat-treated extract for 20 min.(C) Western analysis of RBC infected with EPEC and exposed to either buffer, extract, heat-treated extract, heat-treated extract plus 5 mM CaCl2, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of RBC infected with EPEC or EPECΔeae and treated with either DMEM or extract for 20 min, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.

Mentions: The observation that exposure of infected RBC to low Ca++ solutions, such as buffer or PBS, induces an increase in Tir in the overlying solution, but not the TX-100 soluble fraction, suggests that insertion of Tir into the RBC membrane following exposure to extract, or anchoring of inserted Tir, occurs by a mechanism that does not depend on Ca++. We reasoned that factors from both the extract and the bacteria might contribute to insertion, anchoring or both. To begin to characterize factors within the extract other than calcium, extract was heated to 65 °C for 15 min, or 90 °C for five min, and centrifuged to remove precipitated material prior to incubation with RBC. Heat-treated extract never induced the dramatic increase in Tir in the TX-100 soluble fraction seen following exposure to untreated extract (Figure 8A). Moreover, exposure of infected RBC to heat-treated extract significantly increased the levels of Tir in the overlying solution (Figure 8A), a phenomenon not seen with untreated extract. Microscopy of infected RBC exposed to heat-treated extract also revealed no significant increase in Tir localization beneath attached EPEC compared to treatment with DMEM (Figure 8B). Addition of 5 mM Ca++ to heat-treated extract (resulting in ∼2 mM free Ca++) reduced secretion of Tir into the overlying solution and significantly reduced levels of Tir in the TX-100 soluble fraction (Figure 8C). Taken together, these data indicate that the low Ca++ concentration in extract induces secretion of Tir, but that a heat-labile factor(s) in the extract facilitates efficient insertion or anchoring of Tir in the RBC membrane.


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

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

A Heat-Labile Cytosolic Factor(s) and Intimin Facilitate Translocation of Tir into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, extract, or heat-treated extract that was incubated at (65 °C for 15 min (Extract 65°) or incubated at 90 °C for 5 min (Extract 90°)), or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining after exposure to either DMEM, buffer, extract, or heat-treated extract for 20 min.(C) Western analysis of RBC infected with EPEC and exposed to either buffer, extract, heat-treated extract, heat-treated extract plus 5 mM CaCl2, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of RBC infected with EPEC or EPECΔeae and treated with either DMEM or extract for 20 min, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.
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Related In: Results  -  Collection

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ppat-0040004-g008: A Heat-Labile Cytosolic Factor(s) and Intimin Facilitate Translocation of Tir into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, extract, or heat-treated extract that was incubated at (65 °C for 15 min (Extract 65°) or incubated at 90 °C for 5 min (Extract 90°)), or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining after exposure to either DMEM, buffer, extract, or heat-treated extract for 20 min.(C) Western analysis of RBC infected with EPEC and exposed to either buffer, extract, heat-treated extract, heat-treated extract plus 5 mM CaCl2, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of RBC infected with EPEC or EPECΔeae and treated with either DMEM or extract for 20 min, or left untreated and lysed immediately after infection. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.
Mentions: The observation that exposure of infected RBC to low Ca++ solutions, such as buffer or PBS, induces an increase in Tir in the overlying solution, but not the TX-100 soluble fraction, suggests that insertion of Tir into the RBC membrane following exposure to extract, or anchoring of inserted Tir, occurs by a mechanism that does not depend on Ca++. We reasoned that factors from both the extract and the bacteria might contribute to insertion, anchoring or both. To begin to characterize factors within the extract other than calcium, extract was heated to 65 °C for 15 min, or 90 °C for five min, and centrifuged to remove precipitated material prior to incubation with RBC. Heat-treated extract never induced the dramatic increase in Tir in the TX-100 soluble fraction seen following exposure to untreated extract (Figure 8A). Moreover, exposure of infected RBC to heat-treated extract significantly increased the levels of Tir in the overlying solution (Figure 8A), a phenomenon not seen with untreated extract. Microscopy of infected RBC exposed to heat-treated extract also revealed no significant increase in Tir localization beneath attached EPEC compared to treatment with DMEM (Figure 8B). Addition of 5 mM Ca++ to heat-treated extract (resulting in ∼2 mM free Ca++) reduced secretion of Tir into the overlying solution and significantly reduced levels of Tir in the TX-100 soluble fraction (Figure 8C). Taken together, these data indicate that the low Ca++ concentration in extract induces secretion of Tir, but that a heat-labile factor(s) in the extract facilitates efficient insertion or anchoring of Tir in the RBC membrane.

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