Limits...
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.

Show MeSH

Related in: MedlinePlus

TarP-LD signaling to actin is dependent on the Arp2/3 activity.A, the LD-mediated recruitment of actin required a functional Arp2/3 complex. FAK+/+ MEFs expressing TirM-LD were treated with 100 μm concentrations of the cell-permeable inhibitor CK-666 or the inactive control CK-689 during infection with Δtir EPEC. Actin (green) and p34Arc (red) were visualized with phalloidin or an anti-p34Arc antibody, respectively. Bacteria were visualized by DAPI. White arrowheads indicate colocalization or lack thereof. Note the absence of F-actin aggregates underneath the bacteria on cells exposed to CK-666. The insets show a magnification of a selected area of the cell. Scale bars, 10 μm. Adhered EPEC colocalizing with actin (B) or p34Arc (C) were enumerated, and data are presented as box and whisker plots. Data were compiled from three independent experiments. A range of 680–840 particles was counted for each group. The asterisk indicates statistical significance (ANOVA; actin, p < 0.005; p34Arc, p < 0.3 and post hoc testing Tukey-Kramer; α = 0.01, q = 3.96). D, HeLa cells pretreated with CK-666 or CK-689 were infected with GPIC. Infection was allowed to proceed up to 10-, 30-, and 60-min post-temperature shift. Data were from a minimum of 100 cells from three independent experiments and expressed as a box and whisker plot (see “Experimental Procedures” for details on statistical analysis; ANOVA p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215226&req=5

Figure 8: TarP-LD signaling to actin is dependent on the Arp2/3 activity.A, the LD-mediated recruitment of actin required a functional Arp2/3 complex. FAK+/+ MEFs expressing TirM-LD were treated with 100 μm concentrations of the cell-permeable inhibitor CK-666 or the inactive control CK-689 during infection with Δtir EPEC. Actin (green) and p34Arc (red) were visualized with phalloidin or an anti-p34Arc antibody, respectively. Bacteria were visualized by DAPI. White arrowheads indicate colocalization or lack thereof. Note the absence of F-actin aggregates underneath the bacteria on cells exposed to CK-666. The insets show a magnification of a selected area of the cell. Scale bars, 10 μm. Adhered EPEC colocalizing with actin (B) or p34Arc (C) were enumerated, and data are presented as box and whisker plots. Data were compiled from three independent experiments. A range of 680–840 particles was counted for each group. The asterisk indicates statistical significance (ANOVA; actin, p < 0.005; p34Arc, p < 0.3 and post hoc testing Tukey-Kramer; α = 0.01, q = 3.96). D, HeLa cells pretreated with CK-666 or CK-689 were infected with GPIC. Infection was allowed to proceed up to 10-, 30-, and 60-min post-temperature shift. Data were from a minimum of 100 cells from three independent experiments and expressed as a box and whisker plot (see “Experimental Procedures” for details on statistical analysis; ANOVA p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).

Mentions: To address the potential Arp2/3 requirement, a cell-permeable inhibitor (CK-666) that specifically targeted Arp2/3-mediated actin filament nucleation was used. The inhibitor prevented Arp2/3 from achieving an active conformation by targeting the pocket formed by the subdomain 4 of Arp2 and subdomain 1 of Arp3 without affecting its recruitment to the complex (48). As above, FAK+/+ cells were transfected with the TirM-TarP-LD expression constructs. Recruitment of actin and Arp2/3 were visualized with Alexa 488-phalloidin and anti-p34Arc, respectively. The cells were pretreated with 100 μm concentrations of either CK-666 or the inactive control, CK-689. In cells treated with the CK-689, recruitment of both actin and p34Arc could be seen. In contrast, CK-666 treatment led to the loss of F-actin aggregates (Fig. 8A), indicating that Arp2/3 activation was essential for TarP-LD-mediated actin remodeling. The levels of colocalization of EPEC for either actin or p34Arc were quantified for both CK-666- and CK-689-treated cells. As shown in Fig. 8, B and C, recruitment of actin, but not p34Arc, was significantly reduced in cells treated with CK-666 inhibitor (actin, p < 0.005; p34Arc, p < 0.3, ANOVA, and Tukey-Kramer post hoc test).


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)

TarP-LD signaling to actin is dependent on the Arp2/3 activity.A, the LD-mediated recruitment of actin required a functional Arp2/3 complex. FAK+/+ MEFs expressing TirM-LD were treated with 100 μm concentrations of the cell-permeable inhibitor CK-666 or the inactive control CK-689 during infection with Δtir EPEC. Actin (green) and p34Arc (red) were visualized with phalloidin or an anti-p34Arc antibody, respectively. Bacteria were visualized by DAPI. White arrowheads indicate colocalization or lack thereof. Note the absence of F-actin aggregates underneath the bacteria on cells exposed to CK-666. The insets show a magnification of a selected area of the cell. Scale bars, 10 μm. Adhered EPEC colocalizing with actin (B) or p34Arc (C) were enumerated, and data are presented as box and whisker plots. Data were compiled from three independent experiments. A range of 680–840 particles was counted for each group. The asterisk indicates statistical significance (ANOVA; actin, p < 0.005; p34Arc, p < 0.3 and post hoc testing Tukey-Kramer; α = 0.01, q = 3.96). D, HeLa cells pretreated with CK-666 or CK-689 were infected with GPIC. Infection was allowed to proceed up to 10-, 30-, and 60-min post-temperature shift. Data were from a minimum of 100 cells from three independent experiments and expressed as a box and whisker plot (see “Experimental Procedures” for details on statistical analysis; ANOVA p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215226&req=5

Figure 8: TarP-LD signaling to actin is dependent on the Arp2/3 activity.A, the LD-mediated recruitment of actin required a functional Arp2/3 complex. FAK+/+ MEFs expressing TirM-LD were treated with 100 μm concentrations of the cell-permeable inhibitor CK-666 or the inactive control CK-689 during infection with Δtir EPEC. Actin (green) and p34Arc (red) were visualized with phalloidin or an anti-p34Arc antibody, respectively. Bacteria were visualized by DAPI. White arrowheads indicate colocalization or lack thereof. Note the absence of F-actin aggregates underneath the bacteria on cells exposed to CK-666. The insets show a magnification of a selected area of the cell. Scale bars, 10 μm. Adhered EPEC colocalizing with actin (B) or p34Arc (C) were enumerated, and data are presented as box and whisker plots. Data were compiled from three independent experiments. A range of 680–840 particles was counted for each group. The asterisk indicates statistical significance (ANOVA; actin, p < 0.005; p34Arc, p < 0.3 and post hoc testing Tukey-Kramer; α = 0.01, q = 3.96). D, HeLa cells pretreated with CK-666 or CK-689 were infected with GPIC. Infection was allowed to proceed up to 10-, 30-, and 60-min post-temperature shift. Data were from a minimum of 100 cells from three independent experiments and expressed as a box and whisker plot (see “Experimental Procedures” for details on statistical analysis; ANOVA p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.7).
Mentions: To address the potential Arp2/3 requirement, a cell-permeable inhibitor (CK-666) that specifically targeted Arp2/3-mediated actin filament nucleation was used. The inhibitor prevented Arp2/3 from achieving an active conformation by targeting the pocket formed by the subdomain 4 of Arp2 and subdomain 1 of Arp3 without affecting its recruitment to the complex (48). As above, FAK+/+ cells were transfected with the TirM-TarP-LD expression constructs. Recruitment of actin and Arp2/3 were visualized with Alexa 488-phalloidin and anti-p34Arc, respectively. The cells were pretreated with 100 μm concentrations of either CK-666 or the inactive control, CK-689. In cells treated with the CK-689, recruitment of both actin and p34Arc could be seen. In contrast, CK-666 treatment led to the loss of F-actin aggregates (Fig. 8A), indicating that Arp2/3 activation was essential for TarP-LD-mediated actin remodeling. The levels of colocalization of EPEC for either actin or p34Arc were quantified for both CK-666- and CK-689-treated cells. As shown in Fig. 8, B and C, recruitment of actin, but not p34Arc, was significantly reduced in cells treated with CK-666 inhibitor (actin, p < 0.005; p34Arc, p < 0.3, ANOVA, and Tukey-Kramer post hoc test).

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