Limits...
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.

Show MeSH

Related in: MedlinePlus

TarP orthologs harbor multiple vinculin binding sites. (A) Schematic of TarP orthologs from C. trachomatis serovar L2 (L2), C. trachomatis serovar D (D), C. trachomatis serovar A (A), C. caviae (GPIC), C. abortus (Cab), C. felis (CFe), and C. muridarum (MoPN). Indicated are the locations of the tyrosine-rich phosphorylation domain (orange box), the proline rich domain (green box), actin binding domains (red box), and the LD domain (blue box). Vinculin Binding Site 3 (VBS3; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box) together comprise the Vinculin Binding Domain (VBD). (B) ClustalW sequence alignment of the putative VBS motifs. The numbers indicate the amino acid residue of the amino terminus or carboxy terminus. The consensus sequences shown are based on homology greater than 50%. A 19-residue consensus motif (LLxAAKAVADAxSKLLKAx) was generated by aligning by aligning the 11 VBSs of talin, the three VBSs of IpaA, and the two VBSs of sca4 for comparison to the TarP LD consensus (LxxAAxNVTxxLSxxxxxx). Identical amino acids are in red. Similar residues are in blue. “x” indicates any amino acid.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663276&req=5

Figure 2: TarP orthologs harbor multiple vinculin binding sites. (A) Schematic of TarP orthologs from C. trachomatis serovar L2 (L2), C. trachomatis serovar D (D), C. trachomatis serovar A (A), C. caviae (GPIC), C. abortus (Cab), C. felis (CFe), and C. muridarum (MoPN). Indicated are the locations of the tyrosine-rich phosphorylation domain (orange box), the proline rich domain (green box), actin binding domains (red box), and the LD domain (blue box). Vinculin Binding Site 3 (VBS3; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box) together comprise the Vinculin Binding Domain (VBD). (B) ClustalW sequence alignment of the putative VBS motifs. The numbers indicate the amino acid residue of the amino terminus or carboxy terminus. The consensus sequences shown are based on homology greater than 50%. A 19-residue consensus motif (LLxAAKAVADAxSKLLKAx) was generated by aligning by aligning the 11 VBSs of talin, the three VBSs of IpaA, and the two VBSs of sca4 for comparison to the TarP LD consensus (LxxAAxNVTxxLSxxxxxx). Identical amino acids are in red. Similar residues are in blue. “x” indicates any amino acid.

Mentions: Some proteins that interact with vinculin, including the focal adhesion-associated talin, require VBS consisting of a 19-residue consensus motif (LLxAAKAVADAxSKLLKAx). This consensus sequence was determined from the alignment of the 11 VBSs of talin (Gingras et al., 2005), the three VBSs of IpaA (Park et al., 2011b) and the two VBSs of sca4 (Park et al., 2011a). We hypothesized that the robust vinculin recruitment may involve the virulence factor TarP due to its established function as a signaling scaffold (Lane et al., 2008). Through bioinformatics analyses, candidate VBS motifs were identified in the TarP orthologs of all chlamydial species investigated (Figure 2A). Together, these VBSs comprised the VBD, with VBS1 being the most C-terminal (Figure 2B).


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)

TarP orthologs harbor multiple vinculin binding sites. (A) Schematic of TarP orthologs from C. trachomatis serovar L2 (L2), C. trachomatis serovar D (D), C. trachomatis serovar A (A), C. caviae (GPIC), C. abortus (Cab), C. felis (CFe), and C. muridarum (MoPN). Indicated are the locations of the tyrosine-rich phosphorylation domain (orange box), the proline rich domain (green box), actin binding domains (red box), and the LD domain (blue box). Vinculin Binding Site 3 (VBS3; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box) together comprise the Vinculin Binding Domain (VBD). (B) ClustalW sequence alignment of the putative VBS motifs. The numbers indicate the amino acid residue of the amino terminus or carboxy terminus. The consensus sequences shown are based on homology greater than 50%. A 19-residue consensus motif (LLxAAKAVADAxSKLLKAx) was generated by aligning by aligning the 11 VBSs of talin, the three VBSs of IpaA, and the two VBSs of sca4 for comparison to the TarP LD consensus (LxxAAxNVTxxLSxxxxxx). Identical amino acids are in red. Similar residues are in blue. “x” indicates any amino acid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663276&req=5

Figure 2: TarP orthologs harbor multiple vinculin binding sites. (A) Schematic of TarP orthologs from C. trachomatis serovar L2 (L2), C. trachomatis serovar D (D), C. trachomatis serovar A (A), C. caviae (GPIC), C. abortus (Cab), C. felis (CFe), and C. muridarum (MoPN). Indicated are the locations of the tyrosine-rich phosphorylation domain (orange box), the proline rich domain (green box), actin binding domains (red box), and the LD domain (blue box). Vinculin Binding Site 3 (VBS3; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box) together comprise the Vinculin Binding Domain (VBD). (B) ClustalW sequence alignment of the putative VBS motifs. The numbers indicate the amino acid residue of the amino terminus or carboxy terminus. The consensus sequences shown are based on homology greater than 50%. A 19-residue consensus motif (LLxAAKAVADAxSKLLKAx) was generated by aligning by aligning the 11 VBSs of talin, the three VBSs of IpaA, and the two VBSs of sca4 for comparison to the TarP LD consensus (LxxAAxNVTxxLSxxxxxx). Identical amino acids are in red. Similar residues are in blue. “x” indicates any amino acid.
Mentions: Some proteins that interact with vinculin, including the focal adhesion-associated talin, require VBS consisting of a 19-residue consensus motif (LLxAAKAVADAxSKLLKAx). This consensus sequence was determined from the alignment of the 11 VBSs of talin (Gingras et al., 2005), the three VBSs of IpaA (Park et al., 2011b) and the two VBSs of sca4 (Park et al., 2011a). We hypothesized that the robust vinculin recruitment may involve the virulence factor TarP due to its established function as a signaling scaffold (Lane et al., 2008). Through bioinformatics analyses, candidate VBS motifs were identified in the TarP orthologs of all chlamydial species investigated (Figure 2A). Together, these VBSs comprised the VBD, with VBS1 being the most C-terminal (Figure 2B).

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