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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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EPEC Tir Undergoes Modification in RBC Membranes and Is Rapidly Phosphorylated on Y474 upon Exposure to Extract(A) Western analysis using anti-Tir antibody of lysed EPEC (lane 1) or the TX-100 soluble fractions of RBC infected with EPEC and left untreated (lane 2), or treated with buffer or extract for 30 min (lanes 3 and 4). Note that the molecular weight of Tir increases from 78 kDa (*) (untranslocated Tir) to ∼82 kDa (**) upon infection of RBC, regardless of any post-infection treatment. Also note the increase in the amount of Tir in the TX-100 soluble fraction of infected RBC exposed to extract.(B) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of 3T3 cells infected with EPEC (lane1), or RBC infected with EPEC and exposed to extract for 20 min (lane 2). Note that Tir from RBC infected with EPEC and exposed to extract is tyrosine phosphorylated, but does not undergo the same shift in molecular weight seen in infected 3T3 cells.(C) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody in the TX-100 soluble fraction of either uninfected (lanes 1 and 2) or infected (lanes 3–8) RBC exposed to extract or buffer for 1, 5, or 15 min. Note that Tir is rapidly phosphorylated on the 82 kDa species after exposure to extract (**). A tyrosine phosphorylated protein of approximately the same molecular weight as Tir was evident following infection of RBC and exposure to buffer (*), but further analysis determined that this phosphoprotein was not Tir.(D) Images of RBC infected with EPECΔtir mutants overexpressing either WT Tir (Δtir + pTir ) or Y474F Tir (Δtir + pTirY474F) and exposed to extract for 20 min. 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 the lack of actin polymerization and PY staining in RBC infected with EPECΔtir + pTirY474F. Scale bars represent 10 μm.(E) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of RBC infected with EPECΔtir + pTir or EPECΔtir + pTirY474F and exposed to extract. Note that no phosphorylation of Tir is evident in RBC infected with EPEC Δtir + pTirY474F.
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ppat-0040004-g004: EPEC Tir Undergoes Modification in RBC Membranes and Is Rapidly Phosphorylated on Y474 upon Exposure to Extract(A) Western analysis using anti-Tir antibody of lysed EPEC (lane 1) or the TX-100 soluble fractions of RBC infected with EPEC and left untreated (lane 2), or treated with buffer or extract for 30 min (lanes 3 and 4). Note that the molecular weight of Tir increases from 78 kDa (*) (untranslocated Tir) to ∼82 kDa (**) upon infection of RBC, regardless of any post-infection treatment. Also note the increase in the amount of Tir in the TX-100 soluble fraction of infected RBC exposed to extract.(B) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of 3T3 cells infected with EPEC (lane1), or RBC infected with EPEC and exposed to extract for 20 min (lane 2). Note that Tir from RBC infected with EPEC and exposed to extract is tyrosine phosphorylated, but does not undergo the same shift in molecular weight seen in infected 3T3 cells.(C) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody in the TX-100 soluble fraction of either uninfected (lanes 1 and 2) or infected (lanes 3–8) RBC exposed to extract or buffer for 1, 5, or 15 min. Note that Tir is rapidly phosphorylated on the 82 kDa species after exposure to extract (**). A tyrosine phosphorylated protein of approximately the same molecular weight as Tir was evident following infection of RBC and exposure to buffer (*), but further analysis determined that this phosphoprotein was not Tir.(D) Images of RBC infected with EPECΔtir mutants overexpressing either WT Tir (Δtir + pTir ) or Y474F Tir (Δtir + pTirY474F) and exposed to extract for 20 min. 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 the lack of actin polymerization and PY staining in RBC infected with EPECΔtir + pTirY474F. Scale bars represent 10 μm.(E) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of RBC infected with EPECΔtir + pTir or EPECΔtir + pTirY474F and exposed to extract. Note that no phosphorylation of Tir is evident in RBC infected with EPEC Δtir + pTirY474F.

Mentions: We next determined whether Tir underwent any changes in molecular mass following infection of RBC and exposure to extract. To do this, RBC were infected with EPEC and exposed to buffer or extract, or left untreated, and the Triton X-100 (TX-100) soluble fraction was subjected to SDS-PAGE and western analysis with anti-Tir antibody to assess membrane-associated Tir. Lysates from overnight cultures of EPEC, or TX-100 soluble fractions from HeLa cells previously infected with EPEC were prepared as controls. In samples that were not treated with extract or buffer, but were washed and lysed immediately after infection, we noted a slight increase in the molecular mass of Tir upon infection of RBC compared to Tir in EPEC lysates from overnight cultures (Figure 4A, lane 1 and lane 2). The increase in molecular mass was small, from ∼78 kDa to ∼82 kDa (Figure 4A), but was consistently observed. When infected RBC were exposed to extract, an apparent increase in the amount of Tir present in the TX-100 soluble fraction was observed, but no additional increase in molecular mass was evident (Figure 4A, lane 4). Tir from infected RBC exposed to buffer alone also did not show any additional changes and ran at the same molecular mass as untreated, infected RBC (Figure 4A, lane 3). Notably, the change in molecular mass of Tir induced upon infection of RBC did not correspond to that observed in 3T3 cells infected with EPEC (Figure 4B, bottom panel; see also [29]). These results indicate that Tir undergoes partial host modification upon translocation into the RBC membrane and that this partially modified Tir is sufficient to support actin polymerization in the presence of extract.


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

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

EPEC Tir Undergoes Modification in RBC Membranes and Is Rapidly Phosphorylated on Y474 upon Exposure to Extract(A) Western analysis using anti-Tir antibody of lysed EPEC (lane 1) or the TX-100 soluble fractions of RBC infected with EPEC and left untreated (lane 2), or treated with buffer or extract for 30 min (lanes 3 and 4). Note that the molecular weight of Tir increases from 78 kDa (*) (untranslocated Tir) to ∼82 kDa (**) upon infection of RBC, regardless of any post-infection treatment. Also note the increase in the amount of Tir in the TX-100 soluble fraction of infected RBC exposed to extract.(B) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of 3T3 cells infected with EPEC (lane1), or RBC infected with EPEC and exposed to extract for 20 min (lane 2). Note that Tir from RBC infected with EPEC and exposed to extract is tyrosine phosphorylated, but does not undergo the same shift in molecular weight seen in infected 3T3 cells.(C) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody in the TX-100 soluble fraction of either uninfected (lanes 1 and 2) or infected (lanes 3–8) RBC exposed to extract or buffer for 1, 5, or 15 min. Note that Tir is rapidly phosphorylated on the 82 kDa species after exposure to extract (**). A tyrosine phosphorylated protein of approximately the same molecular weight as Tir was evident following infection of RBC and exposure to buffer (*), but further analysis determined that this phosphoprotein was not Tir.(D) Images of RBC infected with EPECΔtir mutants overexpressing either WT Tir (Δtir + pTir ) or Y474F Tir (Δtir + pTirY474F) and exposed to extract for 20 min. 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 the lack of actin polymerization and PY staining in RBC infected with EPECΔtir + pTirY474F. Scale bars represent 10 μm.(E) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of RBC infected with EPECΔtir + pTir or EPECΔtir + pTirY474F and exposed to extract. Note that no phosphorylation of Tir is evident in RBC infected with EPEC Δtir + pTirY474F.
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Related In: Results  -  Collection

Show All Figures
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ppat-0040004-g004: EPEC Tir Undergoes Modification in RBC Membranes and Is Rapidly Phosphorylated on Y474 upon Exposure to Extract(A) Western analysis using anti-Tir antibody of lysed EPEC (lane 1) or the TX-100 soluble fractions of RBC infected with EPEC and left untreated (lane 2), or treated with buffer or extract for 30 min (lanes 3 and 4). Note that the molecular weight of Tir increases from 78 kDa (*) (untranslocated Tir) to ∼82 kDa (**) upon infection of RBC, regardless of any post-infection treatment. Also note the increase in the amount of Tir in the TX-100 soluble fraction of infected RBC exposed to extract.(B) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of 3T3 cells infected with EPEC (lane1), or RBC infected with EPEC and exposed to extract for 20 min (lane 2). Note that Tir from RBC infected with EPEC and exposed to extract is tyrosine phosphorylated, but does not undergo the same shift in molecular weight seen in infected 3T3 cells.(C) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody in the TX-100 soluble fraction of either uninfected (lanes 1 and 2) or infected (lanes 3–8) RBC exposed to extract or buffer for 1, 5, or 15 min. Note that Tir is rapidly phosphorylated on the 82 kDa species after exposure to extract (**). A tyrosine phosphorylated protein of approximately the same molecular weight as Tir was evident following infection of RBC and exposure to buffer (*), but further analysis determined that this phosphoprotein was not Tir.(D) Images of RBC infected with EPECΔtir mutants overexpressing either WT Tir (Δtir + pTir ) or Y474F Tir (Δtir + pTirY474F) and exposed to extract for 20 min. 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 the lack of actin polymerization and PY staining in RBC infected with EPECΔtir + pTirY474F. Scale bars represent 10 μm.(E) Western analysis using anti-Tir antibody and anti-PY 4G10 antibody of the TX-100 soluble fraction of RBC infected with EPECΔtir + pTir or EPECΔtir + pTirY474F and exposed to extract. Note that no phosphorylation of Tir is evident in RBC infected with EPEC Δtir + pTirY474F.
Mentions: We next determined whether Tir underwent any changes in molecular mass following infection of RBC and exposure to extract. To do this, RBC were infected with EPEC and exposed to buffer or extract, or left untreated, and the Triton X-100 (TX-100) soluble fraction was subjected to SDS-PAGE and western analysis with anti-Tir antibody to assess membrane-associated Tir. Lysates from overnight cultures of EPEC, or TX-100 soluble fractions from HeLa cells previously infected with EPEC were prepared as controls. In samples that were not treated with extract or buffer, but were washed and lysed immediately after infection, we noted a slight increase in the molecular mass of Tir upon infection of RBC compared to Tir in EPEC lysates from overnight cultures (Figure 4A, lane 1 and lane 2). The increase in molecular mass was small, from ∼78 kDa to ∼82 kDa (Figure 4A), but was consistently observed. When infected RBC were exposed to extract, an apparent increase in the amount of Tir present in the TX-100 soluble fraction was observed, but no additional increase in molecular mass was evident (Figure 4A, lane 4). Tir from infected RBC exposed to buffer alone also did not show any additional changes and ran at the same molecular mass as untreated, infected RBC (Figure 4A, lane 3). Notably, the change in molecular mass of Tir induced upon infection of RBC did not correspond to that observed in 3T3 cells infected with EPEC (Figure 4B, bottom panel; see also [29]). These results indicate that Tir undergoes partial host modification upon translocation into the RBC membrane and that this partially modified Tir is sufficient to support actin polymerization in the presence of extract.

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