Limits...
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.

Show MeSH

Related in: MedlinePlus

Tyrosine Kinases Complexed with Cellular Factors Are Required for Phosphorylation of Tir(A) Western analysis of RBC infected with EPEC, or EPECΔtir+pTir, and exposed to DMEM (supplemented with ATP and TX-100) with or without the addition of purified Abl-FL, Abl-p45, or Abl-T315I kinase (2 μl) for 20 min at 37 °C. Samples were blotted and probed with anti-PY 4G10 antibody and then stripped and reprobed with anti-Tir antibody.(B) Western analysis of RBC infected with either EPEC-WT or EPECΔtir and exposed to buffer containing increasing quantities of purified Abl-FL kinase (0–3 μl) for 20 min at 37 °C. The solution overlying the cells (Overlay) was collected to assess phosphorylation of secreted Tir and samples were blotted as in (A).(C) Western analysis of TX-100 soluble fraction of RBC infected either with EPEC, EPECΔtir+pTir, or EPECΔtir+pTiry474F and incubated with extract alone, with extract supplemented with 10 μl PD-166326, or additionally with 2 μl Abl-T315I for 20 min at 37 °C. Samples were blotted as in (A).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211550&req=5

ppat-0040004-g009: Tyrosine Kinases Complexed with Cellular Factors Are Required for Phosphorylation of Tir(A) Western analysis of RBC infected with EPEC, or EPECΔtir+pTir, and exposed to DMEM (supplemented with ATP and TX-100) with or without the addition of purified Abl-FL, Abl-p45, or Abl-T315I kinase (2 μl) for 20 min at 37 °C. Samples were blotted and probed with anti-PY 4G10 antibody and then stripped and reprobed with anti-Tir antibody.(B) Western analysis of RBC infected with either EPEC-WT or EPECΔtir and exposed to buffer containing increasing quantities of purified Abl-FL kinase (0–3 μl) for 20 min at 37 °C. The solution overlying the cells (Overlay) was collected to assess phosphorylation of secreted Tir and samples were blotted as in (A).(C) Western analysis of TX-100 soluble fraction of RBC infected either with EPEC, EPECΔtir+pTir, or EPECΔtir+pTiry474F and incubated with extract alone, with extract supplemented with 10 μl PD-166326, or additionally with 2 μl Abl-T315I for 20 min at 37 °C. Samples were blotted as in (A).

Mentions: Previous reports indicate that both Abl and Src are capable of phosphorylating Tir isolated by immunoprecipitation from intact cells, and that the Abl- and Etk-family kinases are sufficient for Tir phosphorylation in intact cells [10] We next determined whether Abl alone was sufficient to phosphorylate Tir localized in the RBC plasma membrane. To do this, purified active full-length Abl kinase lacking only the first N-terminal 27 amino acid residues (Abl-FL), or truncated active form containing only the SH2 and catalytic domains (Abl-p45), was added to DMEM supplemented with ATP and TX-100, and incubated with infected RBC. Kinase assays were conducted in tandem in the same buffer to assess the capacity of each kinase to phosphorylate GST-Crk, a known Abl substrate. As seen in Figure 9A (left panel), western analysis indicated that no tyrosine phosphorylation of Tir was evident in the TX-100 soluble fractions of samples following addition of either Abl-FL or Abl-p45 at 2 μl per reaction, even when the blot was exposed to film overnight. However, as little as 0.2 μl μlof either kinase readily phosphorylated GST-Crk under the same reaction conditions, indicating that the kinases were active (data not shown). To determine if the lack of Tir phosphorylation following exposure to purified Abl was simply due to low levels of Tir found in the RBC membrane following exposure to DMEM, RBC were infected with EPECΔtir+pTir, and incubated with DMEM plus Abl-FL or Abl-p45 (Figure 9A, right panel). Infection with EPECΔtir+pTir results in high levels of Tir in the TX-100 soluble fraction of infected cells compared to infections with wild-type EPEC. Nevertheless, no Tir phosphorylation could be detected following exposure to DMEM plus the purified kinases compared to DMEM alone. When purified Abl-FL was added to buffer, instead of DMEM, and layered over infected RBC, thus inducing secretion of large amounts of Tir into the overlying solution, phosphorylation of Tir was readily evident in the overlay fraction following addition of as little as 0.5 μl of kinase (Figure 9B). No phosphorylation was evident under these conditions when RBC were infected with EPECΔtir. Together these data suggest that although purified Abl can readily phosphorylate Tir that has been secreted into the overlying solution, phosphorylation sites on Tir within the RBC membrane are not readily accessible to exogenous kinase under these conditions.


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

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

Tyrosine Kinases Complexed with Cellular Factors Are Required for Phosphorylation of Tir(A) Western analysis of RBC infected with EPEC, or EPECΔtir+pTir, and exposed to DMEM (supplemented with ATP and TX-100) with or without the addition of purified Abl-FL, Abl-p45, or Abl-T315I kinase (2 μl) for 20 min at 37 °C. Samples were blotted and probed with anti-PY 4G10 antibody and then stripped and reprobed with anti-Tir antibody.(B) Western analysis of RBC infected with either EPEC-WT or EPECΔtir and exposed to buffer containing increasing quantities of purified Abl-FL kinase (0–3 μl) for 20 min at 37 °C. The solution overlying the cells (Overlay) was collected to assess phosphorylation of secreted Tir and samples were blotted as in (A).(C) Western analysis of TX-100 soluble fraction of RBC infected either with EPEC, EPECΔtir+pTir, or EPECΔtir+pTiry474F and incubated with extract alone, with extract supplemented with 10 μl PD-166326, or additionally with 2 μl Abl-T315I for 20 min at 37 °C. Samples were blotted as in (A).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040004-g009: Tyrosine Kinases Complexed with Cellular Factors Are Required for Phosphorylation of Tir(A) Western analysis of RBC infected with EPEC, or EPECΔtir+pTir, and exposed to DMEM (supplemented with ATP and TX-100) with or without the addition of purified Abl-FL, Abl-p45, or Abl-T315I kinase (2 μl) for 20 min at 37 °C. Samples were blotted and probed with anti-PY 4G10 antibody and then stripped and reprobed with anti-Tir antibody.(B) Western analysis of RBC infected with either EPEC-WT or EPECΔtir and exposed to buffer containing increasing quantities of purified Abl-FL kinase (0–3 μl) for 20 min at 37 °C. The solution overlying the cells (Overlay) was collected to assess phosphorylation of secreted Tir and samples were blotted as in (A).(C) Western analysis of TX-100 soluble fraction of RBC infected either with EPEC, EPECΔtir+pTir, or EPECΔtir+pTiry474F and incubated with extract alone, with extract supplemented with 10 μl PD-166326, or additionally with 2 μl Abl-T315I for 20 min at 37 °C. Samples were blotted as in (A).
Mentions: Previous reports indicate that both Abl and Src are capable of phosphorylating Tir isolated by immunoprecipitation from intact cells, and that the Abl- and Etk-family kinases are sufficient for Tir phosphorylation in intact cells [10] We next determined whether Abl alone was sufficient to phosphorylate Tir localized in the RBC plasma membrane. To do this, purified active full-length Abl kinase lacking only the first N-terminal 27 amino acid residues (Abl-FL), or truncated active form containing only the SH2 and catalytic domains (Abl-p45), was added to DMEM supplemented with ATP and TX-100, and incubated with infected RBC. Kinase assays were conducted in tandem in the same buffer to assess the capacity of each kinase to phosphorylate GST-Crk, a known Abl substrate. As seen in Figure 9A (left panel), western analysis indicated that no tyrosine phosphorylation of Tir was evident in the TX-100 soluble fractions of samples following addition of either Abl-FL or Abl-p45 at 2 μl per reaction, even when the blot was exposed to film overnight. However, as little as 0.2 μl μlof either kinase readily phosphorylated GST-Crk under the same reaction conditions, indicating that the kinases were active (data not shown). To determine if the lack of Tir phosphorylation following exposure to purified Abl was simply due to low levels of Tir found in the RBC membrane following exposure to DMEM, RBC were infected with EPECΔtir+pTir, and incubated with DMEM plus Abl-FL or Abl-p45 (Figure 9A, right panel). Infection with EPECΔtir+pTir results in high levels of Tir in the TX-100 soluble fraction of infected cells compared to infections with wild-type EPEC. Nevertheless, no Tir phosphorylation could be detected following exposure to DMEM plus the purified kinases compared to DMEM alone. When purified Abl-FL was added to buffer, instead of DMEM, and layered over infected RBC, thus inducing secretion of large amounts of Tir into the overlying solution, phosphorylation of Tir was readily evident in the overlay fraction following addition of as little as 0.5 μl of kinase (Figure 9B). No phosphorylation was evident under these conditions when RBC were infected with EPECΔtir. Together these data suggest that although purified Abl can readily phosphorylate Tir that has been secreted into the overlying solution, phosphorylation sites on Tir within the RBC membrane are not readily accessible to exogenous kinase under these conditions.

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