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

Extract Facilitates Release of Tir from EPEC and Translocation Into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, buffer, 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.(B) Western analysis of RBC infected with EPEC and exposed to either DMEM for 20 min (lane 1), or to extract for 1, 5, 10, or 20 min (lanes 2–5). Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with GFP-EPEC and exposed to either buffer or extract for 20 min. Samples were probed with anti-GFP antibody to assess bacterial lysis and then stripped and reprobed with anti-Tir antibody. GFP-EPEC lysed in SDS-PAGE sample buffer and probed with anti-GFP antibody were run as a control to verify GFP expression (lane 1). Note that very little GFP is detected in either the overlay or the TX-100 soluble fraction after exposure to either buffer or extract.(D) Images of RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min or left untreated and fixed immediately after infection. Cells were stained with DAPI (blue) to visualize bacteria, anti-Tir antibody (red) and Alexa-488-phalloidin to visualize actin (green). Scale bar in all images represents 5 μm. Note the increase in Tir staining beneath EPEC attached to RBC after exposure to extract.(E) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining in RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min, or left untreated.(F) Quantitative analysis of colocalization of EPEC (DAPI) and RBC (visualized by Alexa-488-phalloidin) to assess the quantity of EPEC attached to RBC immediately following infection (untreated) or after exposure to either DMEM, buffer, or extract for 20 min.(G) Western analysis using anti-Tir antibody of EPEC cultured in DMEM for 6 h in the absence of RBC and then incubated with either DMEM, buffer, or extract for 20 min, or left untreated and lysed immediately. An aliquot of the supernatant was collected to assess Tir secretion (Overlay) and the bacterial pellet was lysed with SDS-PAGE sample buffer to assess bacterial Tir. Note that both buffer and extract induce secretion of Tir into the solution overlying the bacteria when no RBCs are present.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211550&req=5

ppat-0040004-g006: Extract Facilitates Release of Tir from EPEC and Translocation Into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, buffer, 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.(B) Western analysis of RBC infected with EPEC and exposed to either DMEM for 20 min (lane 1), or to extract for 1, 5, 10, or 20 min (lanes 2–5). Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with GFP-EPEC and exposed to either buffer or extract for 20 min. Samples were probed with anti-GFP antibody to assess bacterial lysis and then stripped and reprobed with anti-Tir antibody. GFP-EPEC lysed in SDS-PAGE sample buffer and probed with anti-GFP antibody were run as a control to verify GFP expression (lane 1). Note that very little GFP is detected in either the overlay or the TX-100 soluble fraction after exposure to either buffer or extract.(D) Images of RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min or left untreated and fixed immediately after infection. Cells were stained with DAPI (blue) to visualize bacteria, anti-Tir antibody (red) and Alexa-488-phalloidin to visualize actin (green). Scale bar in all images represents 5 μm. Note the increase in Tir staining beneath EPEC attached to RBC after exposure to extract.(E) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining in RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min, or left untreated.(F) Quantitative analysis of colocalization of EPEC (DAPI) and RBC (visualized by Alexa-488-phalloidin) to assess the quantity of EPEC attached to RBC immediately following infection (untreated) or after exposure to either DMEM, buffer, or extract for 20 min.(G) Western analysis using anti-Tir antibody of EPEC cultured in DMEM for 6 h in the absence of RBC and then incubated with either DMEM, buffer, or extract for 20 min, or left untreated and lysed immediately. An aliquot of the supernatant was collected to assess Tir secretion (Overlay) and the bacterial pellet was lysed with SDS-PAGE sample buffer to assess bacterial Tir. Note that both buffer and extract induce secretion of Tir into the solution overlying the bacteria when no RBCs are present.

Mentions: To investigate these possibilities, RBC monolayers were infected with EPEC, as in previous experiments, and either left untreated, a measure of the baseline level of Tir in the RBC membrane immediately following infection, or treated with DMEM, buffer or extract for 20 min. Following incubation, samples were assessed for both insertion of Tir into the RBC membrane (Figure 6A, TX-100 soluble) as well as secretion of Tir into the solution overlying the cells (Figure 6A, Overlay). Western analysis and densitometry revealed that treatment of the RBC monolayer with DMEM resulted in less than a two-fold increase in the amount of Tir in the TX-100 soluble fraction compared to untreated RBC. By contrast, RBC treated with extract contained as much as eight fold more Tir in the TX-100 soluble fraction compared to that seen in untreated RBC, or RBC incubated with DMEM, and 20 fold more Tir than RBC treated with buffer (Figure 6A). Notably, no Tir was detected in the solution overlying infected RBC upon treatment with DMEM or extract. However, after treatment of infected RBC with buffer alone, large amounts of Tir were evident in the solution overlying the cells with no increase evident in the TX-100 soluble fraction compared to untreated RBC. Analysis of RBC infected with EPEC and exposed to extract for 1 to 20 min indicated that the increase in Tir in the TX-100-soluble fraction became evident within 5 min, and that no Tir was released into the solution overlying the cells at any time point (Figure 6B). Thus, following infection in DMEM, continued exposure to this media induced little increase in Tir in the TX-100 soluble membrane fraction, and no secretion of Tir into the overlying solution. By contrast, exposure of infected RBC to buffer caused secretion of Tir exclusively into the solution overlying the cells, whereas exposure to extract induced rapid translocation and insertion of Tir into the RBC membrane.


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

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

Extract Facilitates Release of Tir from EPEC and Translocation Into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, buffer, 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.(B) Western analysis of RBC infected with EPEC and exposed to either DMEM for 20 min (lane 1), or to extract for 1, 5, 10, or 20 min (lanes 2–5). Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with GFP-EPEC and exposed to either buffer or extract for 20 min. Samples were probed with anti-GFP antibody to assess bacterial lysis and then stripped and reprobed with anti-Tir antibody. GFP-EPEC lysed in SDS-PAGE sample buffer and probed with anti-GFP antibody were run as a control to verify GFP expression (lane 1). Note that very little GFP is detected in either the overlay or the TX-100 soluble fraction after exposure to either buffer or extract.(D) Images of RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min or left untreated and fixed immediately after infection. Cells were stained with DAPI (blue) to visualize bacteria, anti-Tir antibody (red) and Alexa-488-phalloidin to visualize actin (green). Scale bar in all images represents 5 μm. Note the increase in Tir staining beneath EPEC attached to RBC after exposure to extract.(E) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining in RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min, or left untreated.(F) Quantitative analysis of colocalization of EPEC (DAPI) and RBC (visualized by Alexa-488-phalloidin) to assess the quantity of EPEC attached to RBC immediately following infection (untreated) or after exposure to either DMEM, buffer, or extract for 20 min.(G) Western analysis using anti-Tir antibody of EPEC cultured in DMEM for 6 h in the absence of RBC and then incubated with either DMEM, buffer, or extract for 20 min, or left untreated and lysed immediately. An aliquot of the supernatant was collected to assess Tir secretion (Overlay) and the bacterial pellet was lysed with SDS-PAGE sample buffer to assess bacterial Tir. Note that both buffer and extract induce secretion of Tir into the solution overlying the bacteria when no RBCs are present.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040004-g006: Extract Facilitates Release of Tir from EPEC and Translocation Into the RBC Membrane(A) Western analysis of RBC infected with EPEC and exposed to either DMEM, buffer, 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.(B) Western analysis of RBC infected with EPEC and exposed to either DMEM for 20 min (lane 1), or to extract for 1, 5, 10, or 20 min (lanes 2–5). Both the Overlay and TX-100 soluble fractions were probed with anti-Tir antibody.(C) Western analysis of RBC infected with GFP-EPEC and exposed to either buffer or extract for 20 min. Samples were probed with anti-GFP antibody to assess bacterial lysis and then stripped and reprobed with anti-Tir antibody. GFP-EPEC lysed in SDS-PAGE sample buffer and probed with anti-GFP antibody were run as a control to verify GFP expression (lane 1). Note that very little GFP is detected in either the overlay or the TX-100 soluble fraction after exposure to either buffer or extract.(D) Images of RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min or left untreated and fixed immediately after infection. Cells were stained with DAPI (blue) to visualize bacteria, anti-Tir antibody (red) and Alexa-488-phalloidin to visualize actin (green). Scale bar in all images represents 5 μm. Note the increase in Tir staining beneath EPEC attached to RBC after exposure to extract.(E) Quantitative analysis of colocalization of EPEC (DAPI) and Tir staining in RBC infected with EPEC and exposed to either DMEM, buffer, or extract for 20 min, or left untreated.(F) Quantitative analysis of colocalization of EPEC (DAPI) and RBC (visualized by Alexa-488-phalloidin) to assess the quantity of EPEC attached to RBC immediately following infection (untreated) or after exposure to either DMEM, buffer, or extract for 20 min.(G) Western analysis using anti-Tir antibody of EPEC cultured in DMEM for 6 h in the absence of RBC and then incubated with either DMEM, buffer, or extract for 20 min, or left untreated and lysed immediately. An aliquot of the supernatant was collected to assess Tir secretion (Overlay) and the bacterial pellet was lysed with SDS-PAGE sample buffer to assess bacterial Tir. Note that both buffer and extract induce secretion of Tir into the solution overlying the bacteria when no RBCs are present.
Mentions: To investigate these possibilities, RBC monolayers were infected with EPEC, as in previous experiments, and either left untreated, a measure of the baseline level of Tir in the RBC membrane immediately following infection, or treated with DMEM, buffer or extract for 20 min. Following incubation, samples were assessed for both insertion of Tir into the RBC membrane (Figure 6A, TX-100 soluble) as well as secretion of Tir into the solution overlying the cells (Figure 6A, Overlay). Western analysis and densitometry revealed that treatment of the RBC monolayer with DMEM resulted in less than a two-fold increase in the amount of Tir in the TX-100 soluble fraction compared to untreated RBC. By contrast, RBC treated with extract contained as much as eight fold more Tir in the TX-100 soluble fraction compared to that seen in untreated RBC, or RBC incubated with DMEM, and 20 fold more Tir than RBC treated with buffer (Figure 6A). Notably, no Tir was detected in the solution overlying infected RBC upon treatment with DMEM or extract. However, after treatment of infected RBC with buffer alone, large amounts of Tir were evident in the solution overlying the cells with no increase evident in the TX-100 soluble fraction compared to untreated RBC. Analysis of RBC infected with EPEC and exposed to extract for 1 to 20 min indicated that the increase in Tir in the TX-100-soluble fraction became evident within 5 min, and that no Tir was released into the solution overlying the cells at any time point (Figure 6B). Thus, following infection in DMEM, continued exposure to this media induced little increase in Tir in the TX-100 soluble membrane fraction, and no secretion of Tir into the overlying solution. By contrast, exposure of infected RBC to buffer caused secretion of Tir exclusively into the solution overlying the cells, whereas exposure to extract induced rapid translocation and insertion of Tir into 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