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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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Decreased Ca++ Induces Rapid Release of Tir from EPEC(A) Western analysis of RBC infected with EPEC and exposed to either normal DMEM (2 mM Ca++) or DMEM supplemented with 1 to 4 mM EGTA. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Western analysis of RBC infected with EPEC and exposed to either Ca++-free PBS or Ca++-free PBS supplemented with 0.5 to 2 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with EPEC and either left untreated, or exposed to buffer or extract with or without the addition of 5 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of wild-type EPEC (WT) and EPECΔescN cultured in DMEM for 6 h in the absence of RBC and either lysed with SDS-PAGE sample buffer to verify Tir expression (lanes 1 and 2), or exposed to either DMEM + 4 mM EGTA, Ca++ free PBS, buffer or extract to induce Tir secretion (WT; lanes 3–6, ΔescN; lanes 7–10). Both the lysed bacteria and the Overlay fraction were probed with anti-Tir antibody. Note that although EPECΔescN contained large amounts of Tir when lysed, no secreted Tir was evident following exposure to low Ca++ conditions.
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ppat-0040004-g007: Decreased Ca++ Induces Rapid Release of Tir from EPEC(A) Western analysis of RBC infected with EPEC and exposed to either normal DMEM (2 mM Ca++) or DMEM supplemented with 1 to 4 mM EGTA. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Western analysis of RBC infected with EPEC and exposed to either Ca++-free PBS or Ca++-free PBS supplemented with 0.5 to 2 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with EPEC and either left untreated, or exposed to buffer or extract with or without the addition of 5 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of wild-type EPEC (WT) and EPECΔescN cultured in DMEM for 6 h in the absence of RBC and either lysed with SDS-PAGE sample buffer to verify Tir expression (lanes 1 and 2), or exposed to either DMEM + 4 mM EGTA, Ca++ free PBS, buffer or extract to induce Tir secretion (WT; lanes 3–6, ΔescN; lanes 7–10). Both the lysed bacteria and the Overlay fraction were probed with anti-Tir antibody. Note that although EPECΔescN contained large amounts of Tir when lysed, no secreted Tir was evident following exposure to low Ca++ conditions.

Mentions: To test the possibility that a low Ca++ environment could induce rapid secretion of Tir from EPEC that had been cultured in DMEM, RBC were infected with EPEC, washed, and then incubated for 20 min with either normal DMEM, DMEM supplemented with increasing concentrations of the calcium chelator EGTA, or with Ca++-free PBS supplemented with increasing concentrations of free calcium. As shown in Figure 7A, only a small increase in the amount of Tir was evident in the TX-100 soluble fraction, and there was no secretion into the overlying solution, when the RBC monolayer was incubated in normal DMEM. However, addition of DMEM containing 2 to 4 mM EGTA dramatically increased the secretion of Tir into the overlying solution (Figure 7A). By contrast, secreted Tir was evident in the overlying solution after addition of Ca++-free PBS, but secretion was abolished at Ca++ concentrations above 1 mM (Figure 7B). Notably, no significant increase in Tir was evident in the TX-100 soluble fraction under any condition (Figure 7A and 7B), indicating that induction of Tir secretion by low Ca++ did not result in Tir translocation into the RBC membrane. Finally, treatment of infected RBC with either buffer or extract supplemented with excess Ca++ (5 mM) to contain the same free Ca++ concentration as DMEM, caused no secretion of Tir into the overlying solution and no significant increase in the amount of Tir in the TX-100 soluble fraction, similar to results seen with untreated RBC (Figure 7C). In accordance with previous reports [30,32], these data indicate that a low Ca++ environment can facilitate release of Tir from EPEC and demonstrate that Tir secretion is rapidly induced when cultured EPEC are switched from a high to a low Ca++ environment.


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

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

Decreased Ca++ Induces Rapid Release of Tir from EPEC(A) Western analysis of RBC infected with EPEC and exposed to either normal DMEM (2 mM Ca++) or DMEM supplemented with 1 to 4 mM EGTA. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Western analysis of RBC infected with EPEC and exposed to either Ca++-free PBS or Ca++-free PBS supplemented with 0.5 to 2 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with EPEC and either left untreated, or exposed to buffer or extract with or without the addition of 5 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of wild-type EPEC (WT) and EPECΔescN cultured in DMEM for 6 h in the absence of RBC and either lysed with SDS-PAGE sample buffer to verify Tir expression (lanes 1 and 2), or exposed to either DMEM + 4 mM EGTA, Ca++ free PBS, buffer or extract to induce Tir secretion (WT; lanes 3–6, ΔescN; lanes 7–10). Both the lysed bacteria and the Overlay fraction were probed with anti-Tir antibody. Note that although EPECΔescN contained large amounts of Tir when lysed, no secreted Tir was evident following exposure to low Ca++ conditions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211550&req=5

ppat-0040004-g007: Decreased Ca++ Induces Rapid Release of Tir from EPEC(A) Western analysis of RBC infected with EPEC and exposed to either normal DMEM (2 mM Ca++) or DMEM supplemented with 1 to 4 mM EGTA. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(B) Western analysis of RBC infected with EPEC and exposed to either Ca++-free PBS or Ca++-free PBS supplemented with 0.5 to 2 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with EPEC and either left untreated, or exposed to buffer or extract with or without the addition of 5 mM CaCl2. Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(D) Western analysis of wild-type EPEC (WT) and EPECΔescN cultured in DMEM for 6 h in the absence of RBC and either lysed with SDS-PAGE sample buffer to verify Tir expression (lanes 1 and 2), or exposed to either DMEM + 4 mM EGTA, Ca++ free PBS, buffer or extract to induce Tir secretion (WT; lanes 3–6, ΔescN; lanes 7–10). Both the lysed bacteria and the Overlay fraction were probed with anti-Tir antibody. Note that although EPECΔescN contained large amounts of Tir when lysed, no secreted Tir was evident following exposure to low Ca++ conditions.
Mentions: To test the possibility that a low Ca++ environment could induce rapid secretion of Tir from EPEC that had been cultured in DMEM, RBC were infected with EPEC, washed, and then incubated for 20 min with either normal DMEM, DMEM supplemented with increasing concentrations of the calcium chelator EGTA, or with Ca++-free PBS supplemented with increasing concentrations of free calcium. As shown in Figure 7A, only a small increase in the amount of Tir was evident in the TX-100 soluble fraction, and there was no secretion into the overlying solution, when the RBC monolayer was incubated in normal DMEM. However, addition of DMEM containing 2 to 4 mM EGTA dramatically increased the secretion of Tir into the overlying solution (Figure 7A). By contrast, secreted Tir was evident in the overlying solution after addition of Ca++-free PBS, but secretion was abolished at Ca++ concentrations above 1 mM (Figure 7B). Notably, no significant increase in Tir was evident in the TX-100 soluble fraction under any condition (Figure 7A and 7B), indicating that induction of Tir secretion by low Ca++ did not result in Tir translocation into the RBC membrane. Finally, treatment of infected RBC with either buffer or extract supplemented with excess Ca++ (5 mM) to contain the same free Ca++ concentration as DMEM, caused no secretion of Tir into the overlying solution and no significant increase in the amount of Tir in the TX-100 soluble fraction, similar to results seen with untreated RBC (Figure 7C). In accordance with previous reports [30,32], these data indicate that a low Ca++ environment can facilitate release of Tir from EPEC and demonstrate that Tir secretion is rapidly induced when cultured EPEC are switched from a high to a low Ca++ environment.

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