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The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion.

Thwaites T, Nogueira AT, Campeotto I, Silva AP, Grieshaber SS, Carabeo RA - J. Biol. Chem. (2014)

Bottom Line: The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34(Arc), and actin to the plasma membrane.In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction.Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

View Article: PubMed Central - PubMed

Affiliation: From the Centre for Molecular Microbiology and Infection, Imperial College, London SW7 2AZ, United Kingdom, Bacteriology Section, Programme in Microbiology, Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom, and.

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Recruitment of FAK by TarP-LD domain requires conserved leucine residues.A, Cos7 cells transfected with the indicated plasmid were infected with Δtir EPEC. Arrowheads indicate examples of colocalization or lack thereof in the mutant and negative control. Anti-pY397-FAK and anti-HA antibodies were used to visualize pY397-FAK (green) or TarP-LD/mutLD (red), respectively. Bacteria were visualized by DAPI staining (blue). Insets show a magnification of a selected area of the cell. Scale bars, 10 μm. B, adhered EPEC able to recruit pY397-FAK were enumerated, and data are represented as a box and whisker plot. Data compiled from three independent experiments. A range of 650–1300 particles was counted. The asterisk indicates statistical significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.59). C, oligopeptide pulldown using HeLa cell lysates showed that FAK specifically recognizes the TarP-LD motif, whereas amino acid substitutions abrogated interaction. Lane 1, 10% input; lane 2, wild type LD, lane 3, Leu-to-Ala substitution mutant, lane 4, streptavidin beads only.
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Figure 5: Recruitment of FAK by TarP-LD domain requires conserved leucine residues.A, Cos7 cells transfected with the indicated plasmid were infected with Δtir EPEC. Arrowheads indicate examples of colocalization or lack thereof in the mutant and negative control. Anti-pY397-FAK and anti-HA antibodies were used to visualize pY397-FAK (green) or TarP-LD/mutLD (red), respectively. Bacteria were visualized by DAPI staining (blue). Insets show a magnification of a selected area of the cell. Scale bars, 10 μm. B, adhered EPEC able to recruit pY397-FAK were enumerated, and data are represented as a box and whisker plot. Data compiled from three independent experiments. A range of 650–1300 particles was counted. The asterisk indicates statistical significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.59). C, oligopeptide pulldown using HeLa cell lysates showed that FAK specifically recognizes the TarP-LD motif, whereas amino acid substitutions abrogated interaction. Lane 1, 10% input; lane 2, wild type LD, lane 3, Leu-to-Ala substitution mutant, lane 4, streptavidin beads only.

Mentions: The mutant (TarP-mutLD) was evaluated for the recruitment of pY397-FAK in the EPEC system and the co-precipitation of FAK using custom-synthesized oligopeptides with wild type or mutant LD amino acid sequences. Both experiments were performed in parallel with the wild type TarP-LD and paxillin LD2. As shown in the images in Fig. 5A and quantification in Fig. 5B, TarP-mutLD exhibited a low level of recruitment similar to the negative TirM control and significantly different to TarP-LD and paxillin LD2. The importance of the leucine residues was further confirmed in a co-precipitation experiment where the N-terminal biotinylated oligopeptide bait containing the Leu-to-Ala-substituted mutant form failed to pull down FAK from whole cell lysates (Fig. 5C). Lysates were obtained from cells not treated in any way to induce FAK phosphorylation. Together, the data indicated that, similar to paxillin LD2, the binding of TarP-LD to FAK required the conserved leucine residues.


The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion.

Thwaites T, Nogueira AT, Campeotto I, Silva AP, Grieshaber SS, Carabeo RA - J. Biol. Chem. (2014)

Recruitment of FAK by TarP-LD domain requires conserved leucine residues.A, Cos7 cells transfected with the indicated plasmid were infected with Δtir EPEC. Arrowheads indicate examples of colocalization or lack thereof in the mutant and negative control. Anti-pY397-FAK and anti-HA antibodies were used to visualize pY397-FAK (green) or TarP-LD/mutLD (red), respectively. Bacteria were visualized by DAPI staining (blue). Insets show a magnification of a selected area of the cell. Scale bars, 10 μm. B, adhered EPEC able to recruit pY397-FAK were enumerated, and data are represented as a box and whisker plot. Data compiled from three independent experiments. A range of 650–1300 particles was counted. The asterisk indicates statistical significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.59). C, oligopeptide pulldown using HeLa cell lysates showed that FAK specifically recognizes the TarP-LD motif, whereas amino acid substitutions abrogated interaction. Lane 1, 10% input; lane 2, wild type LD, lane 3, Leu-to-Ala substitution mutant, lane 4, streptavidin beads only.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215226&req=5

Figure 5: Recruitment of FAK by TarP-LD domain requires conserved leucine residues.A, Cos7 cells transfected with the indicated plasmid were infected with Δtir EPEC. Arrowheads indicate examples of colocalization or lack thereof in the mutant and negative control. Anti-pY397-FAK and anti-HA antibodies were used to visualize pY397-FAK (green) or TarP-LD/mutLD (red), respectively. Bacteria were visualized by DAPI staining (blue). Insets show a magnification of a selected area of the cell. Scale bars, 10 μm. B, adhered EPEC able to recruit pY397-FAK were enumerated, and data are represented as a box and whisker plot. Data compiled from three independent experiments. A range of 650–1300 particles was counted. The asterisk indicates statistical significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.59). C, oligopeptide pulldown using HeLa cell lysates showed that FAK specifically recognizes the TarP-LD motif, whereas amino acid substitutions abrogated interaction. Lane 1, 10% input; lane 2, wild type LD, lane 3, Leu-to-Ala substitution mutant, lane 4, streptavidin beads only.
Mentions: The mutant (TarP-mutLD) was evaluated for the recruitment of pY397-FAK in the EPEC system and the co-precipitation of FAK using custom-synthesized oligopeptides with wild type or mutant LD amino acid sequences. Both experiments were performed in parallel with the wild type TarP-LD and paxillin LD2. As shown in the images in Fig. 5A and quantification in Fig. 5B, TarP-mutLD exhibited a low level of recruitment similar to the negative TirM control and significantly different to TarP-LD and paxillin LD2. The importance of the leucine residues was further confirmed in a co-precipitation experiment where the N-terminal biotinylated oligopeptide bait containing the Leu-to-Ala-substituted mutant form failed to pull down FAK from whole cell lysates (Fig. 5C). Lysates were obtained from cells not treated in any way to induce FAK phosphorylation. Together, the data indicated that, similar to paxillin LD2, the binding of TarP-LD to FAK required the conserved leucine residues.

Bottom Line: The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34(Arc), and actin to the plasma membrane.In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction.Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

View Article: PubMed Central - PubMed

Affiliation: From the Centre for Molecular Microbiology and Infection, Imperial College, London SW7 2AZ, United Kingdom, Bacteriology Section, Programme in Microbiology, Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom, and.

Show MeSH
Related in: MedlinePlus