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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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FAK transiently localizes to sites of chlamydial adherence and is required for invasion.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-FAK (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar, 5 μm. B, pixel intensities for representative areas of EGFP-FAK recruitment were enumerated and plotted as pixel intensity in arbitrary units (a.u.) over time. C, Cos7 cells were infected with C. caviae (GPIC) EBs (DAPI, blue) and stained with an anti-phospho-Y397 FAK (pY397-FAK; red) antibody. White arrowheads indicate pY397-FAK and GFP-FAK colocalizing with GPIC EBs. Scale bar, 5 μm. D, EB colocalization with phospho-FAK (pY397-FAK), GFP-FAK, or both was enumerated, and data expressed are expressed as as the percentage of total EBs displaying colocalization. The asterisk and bars indicate significant difference between pY397-FAK, GFP-FAK, or both from T = 0 to T = 10 (one way ANOVA, Tukey's post hoc test q = 4.7, α = 0.01, pY397-FAK p < 0.005; GFP-FAK p < 0.004; both p < 0.03). Correlation of the recruitment kinetics was tested using Spearmann's test for correlation (one-tailed; p < 0.005). Data from three independent experiments were represented as the mean ± S.D. E, infection-dependent induction of FAK phosphorylation (Tyr-397). Solubilized HeLa lysates were probed with an anti-phospho-Tyr-397 FAK or anti-FAK antibody to monitor FAK activation or total FAK, respectively. The lane labeled (Uninf 10′) refers to a parallel sample collected at the 10-min time point after mock-infection. β-Tubulin served as a loading control. F, C. caviae GPIC invasion required FAK. Infection of FAK+/+ and FAK−/− cells was allowed to proceed up to a 10-min post-temperature shift. Data are from a minimum of 100 cells from three independent experiments. The asterisk indicates statistical significance (ANOVA p < 0.005. and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).
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Figure 1: FAK transiently localizes to sites of chlamydial adherence and is required for invasion.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-FAK (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar, 5 μm. B, pixel intensities for representative areas of EGFP-FAK recruitment were enumerated and plotted as pixel intensity in arbitrary units (a.u.) over time. C, Cos7 cells were infected with C. caviae (GPIC) EBs (DAPI, blue) and stained with an anti-phospho-Y397 FAK (pY397-FAK; red) antibody. White arrowheads indicate pY397-FAK and GFP-FAK colocalizing with GPIC EBs. Scale bar, 5 μm. D, EB colocalization with phospho-FAK (pY397-FAK), GFP-FAK, or both was enumerated, and data expressed are expressed as as the percentage of total EBs displaying colocalization. The asterisk and bars indicate significant difference between pY397-FAK, GFP-FAK, or both from T = 0 to T = 10 (one way ANOVA, Tukey's post hoc test q = 4.7, α = 0.01, pY397-FAK p < 0.005; GFP-FAK p < 0.004; both p < 0.03). Correlation of the recruitment kinetics was tested using Spearmann's test for correlation (one-tailed; p < 0.005). Data from three independent experiments were represented as the mean ± S.D. E, infection-dependent induction of FAK phosphorylation (Tyr-397). Solubilized HeLa lysates were probed with an anti-phospho-Tyr-397 FAK or anti-FAK antibody to monitor FAK activation or total FAK, respectively. The lane labeled (Uninf 10′) refers to a parallel sample collected at the 10-min time point after mock-infection. β-Tubulin served as a loading control. F, C. caviae GPIC invasion required FAK. Infection of FAK+/+ and FAK−/− cells was allowed to proceed up to a 10-min post-temperature shift. Data are from a minimum of 100 cells from three independent experiments. The asterisk indicates statistical significance (ANOVA p < 0.005. and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).

Mentions: To gain insight into the involvement of FAK in chlamydial invasion, its localization during infection was investigated by confocal microscopy of live and fixed samples. Using live-cell imaging, we observed the immediate and specific localization of EGFP-FAK to the Chlamydia entry sites (Fig. 1A). EGFP-FAK fluorescence intensity at areas of EB attachment was quantified and adjusted relative to the neighboring regions lacking EBs. The data indicated that the EB-localized fluorescence only persisted for an average of 180 ± 60 s (Fig. 1B). This rapid and transient recruitment was consistent with previously published data on the recruitment of Rac GTPase, the Arp3 subunit of the Arp2/3 complex, and actin (13, 14).


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)

FAK transiently localizes to sites of chlamydial adherence and is required for invasion.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-FAK (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar, 5 μm. B, pixel intensities for representative areas of EGFP-FAK recruitment were enumerated and plotted as pixel intensity in arbitrary units (a.u.) over time. C, Cos7 cells were infected with C. caviae (GPIC) EBs (DAPI, blue) and stained with an anti-phospho-Y397 FAK (pY397-FAK; red) antibody. White arrowheads indicate pY397-FAK and GFP-FAK colocalizing with GPIC EBs. Scale bar, 5 μm. D, EB colocalization with phospho-FAK (pY397-FAK), GFP-FAK, or both was enumerated, and data expressed are expressed as as the percentage of total EBs displaying colocalization. The asterisk and bars indicate significant difference between pY397-FAK, GFP-FAK, or both from T = 0 to T = 10 (one way ANOVA, Tukey's post hoc test q = 4.7, α = 0.01, pY397-FAK p < 0.005; GFP-FAK p < 0.004; both p < 0.03). Correlation of the recruitment kinetics was tested using Spearmann's test for correlation (one-tailed; p < 0.005). Data from three independent experiments were represented as the mean ± S.D. E, infection-dependent induction of FAK phosphorylation (Tyr-397). Solubilized HeLa lysates were probed with an anti-phospho-Tyr-397 FAK or anti-FAK antibody to monitor FAK activation or total FAK, respectively. The lane labeled (Uninf 10′) refers to a parallel sample collected at the 10-min time point after mock-infection. β-Tubulin served as a loading control. F, C. caviae GPIC invasion required FAK. Infection of FAK+/+ and FAK−/− cells was allowed to proceed up to a 10-min post-temperature shift. Data are from a minimum of 100 cells from three independent experiments. The asterisk indicates statistical significance (ANOVA p < 0.005. and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).
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Figure 1: FAK transiently localizes to sites of chlamydial adherence and is required for invasion.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-FAK (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar, 5 μm. B, pixel intensities for representative areas of EGFP-FAK recruitment were enumerated and plotted as pixel intensity in arbitrary units (a.u.) over time. C, Cos7 cells were infected with C. caviae (GPIC) EBs (DAPI, blue) and stained with an anti-phospho-Y397 FAK (pY397-FAK; red) antibody. White arrowheads indicate pY397-FAK and GFP-FAK colocalizing with GPIC EBs. Scale bar, 5 μm. D, EB colocalization with phospho-FAK (pY397-FAK), GFP-FAK, or both was enumerated, and data expressed are expressed as as the percentage of total EBs displaying colocalization. The asterisk and bars indicate significant difference between pY397-FAK, GFP-FAK, or both from T = 0 to T = 10 (one way ANOVA, Tukey's post hoc test q = 4.7, α = 0.01, pY397-FAK p < 0.005; GFP-FAK p < 0.004; both p < 0.03). Correlation of the recruitment kinetics was tested using Spearmann's test for correlation (one-tailed; p < 0.005). Data from three independent experiments were represented as the mean ± S.D. E, infection-dependent induction of FAK phosphorylation (Tyr-397). Solubilized HeLa lysates were probed with an anti-phospho-Tyr-397 FAK or anti-FAK antibody to monitor FAK activation or total FAK, respectively. The lane labeled (Uninf 10′) refers to a parallel sample collected at the 10-min time point after mock-infection. β-Tubulin served as a loading control. F, C. caviae GPIC invasion required FAK. Infection of FAK+/+ and FAK−/− cells was allowed to proceed up to a 10-min post-temperature shift. Data are from a minimum of 100 cells from three independent experiments. The asterisk indicates statistical significance (ANOVA p < 0.005. and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).
Mentions: To gain insight into the involvement of FAK in chlamydial invasion, its localization during infection was investigated by confocal microscopy of live and fixed samples. Using live-cell imaging, we observed the immediate and specific localization of EGFP-FAK to the Chlamydia entry sites (Fig. 1A). EGFP-FAK fluorescence intensity at areas of EB attachment was quantified and adjusted relative to the neighboring regions lacking EBs. The data indicated that the EB-localized fluorescence only persisted for an average of 180 ± 60 s (Fig. 1B). This rapid and transient recruitment was consistent with previously published data on the recruitment of Rac GTPase, the Arp3 subunit of the Arp2/3 complex, and actin (13, 14).

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