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Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane.

Thwaites TR, Pedrosa AT, Peacock TP, Carabeo RA - Front Cell Infect Microbiol (2015)

Bottom Line: The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin.As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin.Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

View Article: PubMed Central - PubMed

Affiliation: Programme in Microbiology, Institute of Medical Sciences, University of Aberdeen Aberdeen, UK ; Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London London, UK.

ABSTRACT
The mammalian protein vinculin is often a target of bacterial pathogens to subvert locally host cell actin dynamics. In Chlamydia infection, vinculin has been implicated in RNA interference screens, but the molecular basis for vinculin requirement has not been characterized. In this report, we show that vinculin was involved in the actin recruitment and F-actin assembly at the plasma membrane to facilitate invasion. Vinculin was recruited to the plasma membrane via its interaction with a specific tripartite motif within TarP that resembles the vinculin-binding domain (VBD) found in the Shigella invasion factor IpaA. The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin. In vitro pulldown assays for protein-protein interaction and imaging-based evaluation of recruitment to the plasma membrane demonstrated the essential role of the vinculin-binding site 1 (VBS1), and the dispensability of VBS2 and VBS3. As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin. Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

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Related in: MedlinePlus

Functional evaluation of the individual VBS motif revealed the importance of VBS1. (A) Schematic of Vinculin Binding Domain (TirM-VBD) derivatives designed for use with the EPEC-based assay. Indicated are the locations of the N-terminal membrane targeting sequence (MTS; cyan box), Ha-tag (red box), TirM (amino acids 260–395; purple box), proline rich domain (green box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (orange box), and C-terminal VBS1 (yellow box). Δ indicates amino acids deleted in mutant TarP proteins, and the numbers indicate amino acid positions encoded within the C. caviae TarP gene. Yellow box with black stripes indicates a mutated VBS derivative. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), a VBD mutant derivative in which the critical leucines of VBS1 were converted to serines [TirM-VBS1(mut)-2-3], the VBD deleted for VBS1 (TirM-ΔVBS1), the VBD deleted for VBS2 (TirM-ΔVBS2), the VBD deleted for VBS3 (TirM-ΔVBS3), VBS1 or VBS3 were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of Vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-Vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. (C) Adhered EPEC able to recruit Vinculin were enumerated for VBD, VBS1(mut)-2-3, ΔVBS1, ΔVBS2, ΔVBS3, VBS1, or VBS3, and data represented as box and whisker plot. Data compiled from three independent experiments. The graph shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 200–550 particles was counted. The asterisk and bars indicate significant difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.05). (D) The interaction of vinculin with the VBSs was evaluated by co-immunoprecipitation of HA-tagged VBD derivatives along with the full-length TarP and the negative control TirM at 32 h post-transfection. The co-precipitated vinculin protein and the recombinant TarP and/or VBD constructs were visualized by western blot using antibodies to vinculin and the HA-tag, respectively. The HA-tagged proteins are indicated by asterisks. Note that the loss of VBS1 either through deletion or mutation consistently led to the loss of interaction with vinculin.
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Figure 7: Functional evaluation of the individual VBS motif revealed the importance of VBS1. (A) Schematic of Vinculin Binding Domain (TirM-VBD) derivatives designed for use with the EPEC-based assay. Indicated are the locations of the N-terminal membrane targeting sequence (MTS; cyan box), Ha-tag (red box), TirM (amino acids 260–395; purple box), proline rich domain (green box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (orange box), and C-terminal VBS1 (yellow box). Δ indicates amino acids deleted in mutant TarP proteins, and the numbers indicate amino acid positions encoded within the C. caviae TarP gene. Yellow box with black stripes indicates a mutated VBS derivative. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), a VBD mutant derivative in which the critical leucines of VBS1 were converted to serines [TirM-VBS1(mut)-2-3], the VBD deleted for VBS1 (TirM-ΔVBS1), the VBD deleted for VBS2 (TirM-ΔVBS2), the VBD deleted for VBS3 (TirM-ΔVBS3), VBS1 or VBS3 were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of Vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-Vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. (C) Adhered EPEC able to recruit Vinculin were enumerated for VBD, VBS1(mut)-2-3, ΔVBS1, ΔVBS2, ΔVBS3, VBS1, or VBS3, and data represented as box and whisker plot. Data compiled from three independent experiments. The graph shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 200–550 particles was counted. The asterisk and bars indicate significant difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.05). (D) The interaction of vinculin with the VBSs was evaluated by co-immunoprecipitation of HA-tagged VBD derivatives along with the full-length TarP and the negative control TirM at 32 h post-transfection. The co-precipitated vinculin protein and the recombinant TarP and/or VBD constructs were visualized by western blot using antibodies to vinculin and the HA-tag, respectively. The HA-tagged proteins are indicated by asterisks. Note that the loss of VBS1 either through deletion or mutation consistently led to the loss of interaction with vinculin.

Mentions: The S. flexneri effector IpaA possesses three functional VBSs in tandem arrangement, of which the highest affinity C-terminal motif functioning as the trigger for vinculin activation (Tran Van Nhieu et al., 1997). Like the IpaA effector, C. caviae TarP harbors three VBS motifs. Helical wheel representations of the C. caviae TarP VBS motifs showed VBS1 to be most similar to the C-terminal VBS of IpaA (IpaA-VBS1), with comparable directions and magnitudes of the hydrophobic moments. Conversely, VBS2 or VBS3 have smaller magnitudes of the hydrophobic moments, indicating lower amphiphilicity (Figure S2). Because of the functional differences between the IpaA VBSs, we investigated if the same could be true for TarP VSB1, 2, and 3. Various combinations of VBSs were investigated for vinculin recruitment frequency in the context of the EPEC system (Figure 7A). As shown in Figures 7B,C, deletion of either VBS2 or VBS3 while retaining VBS1 (TirM-ΔVBS2, TirM-ΔVBS3, or TirM-ΔVBS2,3) had minimal influence on the incidence of vinculin recruitment in comparison to the WT VBD, hinting at the greater functional importance of VBS1. This was confirmed through the progressive deletions of the VBD that removed either VBS1 only or VBS1 and VBS2, with both deletion constructs leading to notable decreases in the incidence of vinculin recruitment. Scoring individual EPEC particles for colocalization with vinculin demonstrated that vinculin recruitment frequencies decreased 1.6 and 2.4-fold in cells expressing TirM-ΔVBS1 or TirM-ΔVBS1,2 respectively, relative to WT VBD (Figure 7C; p < 0.03; ANOVA and Tukey-Kramer post-hoc test). Also, a VBD mutant derivative (VBS1mut-2-3) in which the conserved hydrophobic residues of VBS1 were changed to serine was quantitatively and qualitatively similar to ΔVBS1. The data pointed to VBS1 accounting for the majority of VBD function.


Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane.

Thwaites TR, Pedrosa AT, Peacock TP, Carabeo RA - Front Cell Infect Microbiol (2015)

Functional evaluation of the individual VBS motif revealed the importance of VBS1. (A) Schematic of Vinculin Binding Domain (TirM-VBD) derivatives designed for use with the EPEC-based assay. Indicated are the locations of the N-terminal membrane targeting sequence (MTS; cyan box), Ha-tag (red box), TirM (amino acids 260–395; purple box), proline rich domain (green box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (orange box), and C-terminal VBS1 (yellow box). Δ indicates amino acids deleted in mutant TarP proteins, and the numbers indicate amino acid positions encoded within the C. caviae TarP gene. Yellow box with black stripes indicates a mutated VBS derivative. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), a VBD mutant derivative in which the critical leucines of VBS1 were converted to serines [TirM-VBS1(mut)-2-3], the VBD deleted for VBS1 (TirM-ΔVBS1), the VBD deleted for VBS2 (TirM-ΔVBS2), the VBD deleted for VBS3 (TirM-ΔVBS3), VBS1 or VBS3 were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of Vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-Vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. (C) Adhered EPEC able to recruit Vinculin were enumerated for VBD, VBS1(mut)-2-3, ΔVBS1, ΔVBS2, ΔVBS3, VBS1, or VBS3, and data represented as box and whisker plot. Data compiled from three independent experiments. The graph shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 200–550 particles was counted. The asterisk and bars indicate significant difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.05). (D) The interaction of vinculin with the VBSs was evaluated by co-immunoprecipitation of HA-tagged VBD derivatives along with the full-length TarP and the negative control TirM at 32 h post-transfection. The co-precipitated vinculin protein and the recombinant TarP and/or VBD constructs were visualized by western blot using antibodies to vinculin and the HA-tag, respectively. The HA-tagged proteins are indicated by asterisks. Note that the loss of VBS1 either through deletion or mutation consistently led to the loss of interaction with vinculin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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Figure 7: Functional evaluation of the individual VBS motif revealed the importance of VBS1. (A) Schematic of Vinculin Binding Domain (TirM-VBD) derivatives designed for use with the EPEC-based assay. Indicated are the locations of the N-terminal membrane targeting sequence (MTS; cyan box), Ha-tag (red box), TirM (amino acids 260–395; purple box), proline rich domain (green box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (orange box), and C-terminal VBS1 (yellow box). Δ indicates amino acids deleted in mutant TarP proteins, and the numbers indicate amino acid positions encoded within the C. caviae TarP gene. Yellow box with black stripes indicates a mutated VBS derivative. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), a VBD mutant derivative in which the critical leucines of VBS1 were converted to serines [TirM-VBS1(mut)-2-3], the VBD deleted for VBS1 (TirM-ΔVBS1), the VBD deleted for VBS2 (TirM-ΔVBS2), the VBD deleted for VBS3 (TirM-ΔVBS3), VBS1 or VBS3 were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of Vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-Vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. (C) Adhered EPEC able to recruit Vinculin were enumerated for VBD, VBS1(mut)-2-3, ΔVBS1, ΔVBS2, ΔVBS3, VBS1, or VBS3, and data represented as box and whisker plot. Data compiled from three independent experiments. The graph shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 200–550 particles was counted. The asterisk and bars indicate significant difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.05). (D) The interaction of vinculin with the VBSs was evaluated by co-immunoprecipitation of HA-tagged VBD derivatives along with the full-length TarP and the negative control TirM at 32 h post-transfection. The co-precipitated vinculin protein and the recombinant TarP and/or VBD constructs were visualized by western blot using antibodies to vinculin and the HA-tag, respectively. The HA-tagged proteins are indicated by asterisks. Note that the loss of VBS1 either through deletion or mutation consistently led to the loss of interaction with vinculin.
Mentions: The S. flexneri effector IpaA possesses three functional VBSs in tandem arrangement, of which the highest affinity C-terminal motif functioning as the trigger for vinculin activation (Tran Van Nhieu et al., 1997). Like the IpaA effector, C. caviae TarP harbors three VBS motifs. Helical wheel representations of the C. caviae TarP VBS motifs showed VBS1 to be most similar to the C-terminal VBS of IpaA (IpaA-VBS1), with comparable directions and magnitudes of the hydrophobic moments. Conversely, VBS2 or VBS3 have smaller magnitudes of the hydrophobic moments, indicating lower amphiphilicity (Figure S2). Because of the functional differences between the IpaA VBSs, we investigated if the same could be true for TarP VSB1, 2, and 3. Various combinations of VBSs were investigated for vinculin recruitment frequency in the context of the EPEC system (Figure 7A). As shown in Figures 7B,C, deletion of either VBS2 or VBS3 while retaining VBS1 (TirM-ΔVBS2, TirM-ΔVBS3, or TirM-ΔVBS2,3) had minimal influence on the incidence of vinculin recruitment in comparison to the WT VBD, hinting at the greater functional importance of VBS1. This was confirmed through the progressive deletions of the VBD that removed either VBS1 only or VBS1 and VBS2, with both deletion constructs leading to notable decreases in the incidence of vinculin recruitment. Scoring individual EPEC particles for colocalization with vinculin demonstrated that vinculin recruitment frequencies decreased 1.6 and 2.4-fold in cells expressing TirM-ΔVBS1 or TirM-ΔVBS1,2 respectively, relative to WT VBD (Figure 7C; p < 0.03; ANOVA and Tukey-Kramer post-hoc test). Also, a VBD mutant derivative (VBS1mut-2-3) in which the conserved hydrophobic residues of VBS1 were changed to serine was quantitatively and qualitatively similar to ΔVBS1. The data pointed to VBS1 accounting for the majority of VBD function.

Bottom Line: The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin.As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin.Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

View Article: PubMed Central - PubMed

Affiliation: Programme in Microbiology, Institute of Medical Sciences, University of Aberdeen Aberdeen, UK ; Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London London, UK.

ABSTRACT
The mammalian protein vinculin is often a target of bacterial pathogens to subvert locally host cell actin dynamics. In Chlamydia infection, vinculin has been implicated in RNA interference screens, but the molecular basis for vinculin requirement has not been characterized. In this report, we show that vinculin was involved in the actin recruitment and F-actin assembly at the plasma membrane to facilitate invasion. Vinculin was recruited to the plasma membrane via its interaction with a specific tripartite motif within TarP that resembles the vinculin-binding domain (VBD) found in the Shigella invasion factor IpaA. The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin. In vitro pulldown assays for protein-protein interaction and imaging-based evaluation of recruitment to the plasma membrane demonstrated the essential role of the vinculin-binding site 1 (VBS1), and the dispensability of VBS2 and VBS3. As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin. Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

Show MeSH
Related in: MedlinePlus