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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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Cdc42 is important in the TarP-LD- and FAK-dependent recruitment of p34Arc and actin.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-Cdc42 (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar = 5 μm. B, adhered EPEC able to recruit EGFP-Cdc42 were enumerated for the constructs indicated, and data are represented as a box and whisker plot. Approximately 500–800 particles were counted for each construct. The asterisks indicate significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.80). C, representative images analyzed to arrive at the data shown in B with a dotted square border indicating the region represented in the inset images; Inset images are of HA-TirM-TarP derivative (red), EGFP-Cdc42 (green), Δtir EPEC stained with DAPI (blue), and merged. Scale bar, 10 μm. D, EGFP-Cdc42 recruitment requires FAK, as determined using FAK+/+ and FAK−/− MEFs. E, cells expressing the dominant-negative mutant EGFP-Cdc42N17 showed decreased incidence of actin remodeling and p34Arc recruitment at the sites of bacterial adherence. F, representative images analyzed to arrive at the data shown in E with a dotted square border indicating the region represented in the inset images; inset images are of HA-TirM-TarP derivative (red), actin or p34Arc (green), Δtir EPEC stained with DAPI (blue), and merged. An additional inset image (grayscale) is included to demonstrate expression of either EGFP-Cdc42 or EGFP-Cdc42N17 mutant. Scale bar, 10 μm.
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Figure 9: Cdc42 is important in the TarP-LD- and FAK-dependent recruitment of p34Arc and actin.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-Cdc42 (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar = 5 μm. B, adhered EPEC able to recruit EGFP-Cdc42 were enumerated for the constructs indicated, and data are represented as a box and whisker plot. Approximately 500–800 particles were counted for each construct. The asterisks indicate significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.80). C, representative images analyzed to arrive at the data shown in B with a dotted square border indicating the region represented in the inset images; Inset images are of HA-TirM-TarP derivative (red), EGFP-Cdc42 (green), Δtir EPEC stained with DAPI (blue), and merged. Scale bar, 10 μm. D, EGFP-Cdc42 recruitment requires FAK, as determined using FAK+/+ and FAK−/− MEFs. E, cells expressing the dominant-negative mutant EGFP-Cdc42N17 showed decreased incidence of actin remodeling and p34Arc recruitment at the sites of bacterial adherence. F, representative images analyzed to arrive at the data shown in E with a dotted square border indicating the region represented in the inset images; inset images are of HA-TirM-TarP derivative (red), actin or p34Arc (green), Δtir EPEC stained with DAPI (blue), and merged. An additional inset image (grayscale) is included to demonstrate expression of either EGFP-Cdc42 or EGFP-Cdc42N17 mutant. Scale bar, 10 μm.

Mentions: First, the recruitment of Cdc42 reported by Subtil et al. (20) was verified by infecting EGFP-Cdc42-expressing cells with CMTPX-labeled C. caviae strain GPIC and monitoring EGFP-Cdc42 recruitment by live-cell microscopy (Fig. 9A) at 30-s intervals. As with EGFP-FAK, the recruitment was rapid and transient. Having established Cdc42 recruitment during chlamydial invasion, the molecular basis for this event was investigated using the EPEC system described above. Different deletion derivatives of TarP were evaluated for their ability to support the recruitment of EGFP-Cdc42 to the plasma membrane. By enumerating the incidence of recruitment, we found that those constructs that retained recruitment of FAK and actin also were able to recruit Cdc42. Fig. 9B showed that TarP1–714 was able to support Cdc42 recruitment, but progressive deletion (TarP1–639) significantly reduced recruitment incidence to the level of the negative control TirM (3-fold difference versus TirM; p < 0.005). Furthermore, TarP-LD was sufficient to recruit Cdc42 (3-fold difference versus TirM; p < 0.001). Importantly, the conserved Leu residues were essential, with TarP-mutLD displaying a 3-fold decrease in EGFP-Cdc42 recruitment (Fig. 9B). Fig. 9C shows representative images of EGFP-Cdc42 recruitment by select TirM-TarP constructs.


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)

Cdc42 is important in the TarP-LD- and FAK-dependent recruitment of p34Arc and actin.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-Cdc42 (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar = 5 μm. B, adhered EPEC able to recruit EGFP-Cdc42 were enumerated for the constructs indicated, and data are represented as a box and whisker plot. Approximately 500–800 particles were counted for each construct. The asterisks indicate significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.80). C, representative images analyzed to arrive at the data shown in B with a dotted square border indicating the region represented in the inset images; Inset images are of HA-TirM-TarP derivative (red), EGFP-Cdc42 (green), Δtir EPEC stained with DAPI (blue), and merged. Scale bar, 10 μm. D, EGFP-Cdc42 recruitment requires FAK, as determined using FAK+/+ and FAK−/− MEFs. E, cells expressing the dominant-negative mutant EGFP-Cdc42N17 showed decreased incidence of actin remodeling and p34Arc recruitment at the sites of bacterial adherence. F, representative images analyzed to arrive at the data shown in E with a dotted square border indicating the region represented in the inset images; inset images are of HA-TirM-TarP derivative (red), actin or p34Arc (green), Δtir EPEC stained with DAPI (blue), and merged. An additional inset image (grayscale) is included to demonstrate expression of either EGFP-Cdc42 or EGFP-Cdc42N17 mutant. Scale bar, 10 μm.
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Figure 9: Cdc42 is important in the TarP-LD- and FAK-dependent recruitment of p34Arc and actin.A, binding of CMTPX-labeled C. caviae strain GPIC (red) to cells induces a highly localized and transient recruitment of EGFP-Cdc42 (green). The images were obtained by live cell microscopy at 30-s intervals. Scale bar = 5 μm. B, adhered EPEC able to recruit EGFP-Cdc42 were enumerated for the constructs indicated, and data are represented as a box and whisker plot. Approximately 500–800 particles were counted for each construct. The asterisks indicate significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.80). C, representative images analyzed to arrive at the data shown in B with a dotted square border indicating the region represented in the inset images; Inset images are of HA-TirM-TarP derivative (red), EGFP-Cdc42 (green), Δtir EPEC stained with DAPI (blue), and merged. Scale bar, 10 μm. D, EGFP-Cdc42 recruitment requires FAK, as determined using FAK+/+ and FAK−/− MEFs. E, cells expressing the dominant-negative mutant EGFP-Cdc42N17 showed decreased incidence of actin remodeling and p34Arc recruitment at the sites of bacterial adherence. F, representative images analyzed to arrive at the data shown in E with a dotted square border indicating the region represented in the inset images; inset images are of HA-TirM-TarP derivative (red), actin or p34Arc (green), Δtir EPEC stained with DAPI (blue), and merged. An additional inset image (grayscale) is included to demonstrate expression of either EGFP-Cdc42 or EGFP-Cdc42N17 mutant. Scale bar, 10 μm.
Mentions: First, the recruitment of Cdc42 reported by Subtil et al. (20) was verified by infecting EGFP-Cdc42-expressing cells with CMTPX-labeled C. caviae strain GPIC and monitoring EGFP-Cdc42 recruitment by live-cell microscopy (Fig. 9A) at 30-s intervals. As with EGFP-FAK, the recruitment was rapid and transient. Having established Cdc42 recruitment during chlamydial invasion, the molecular basis for this event was investigated using the EPEC system described above. Different deletion derivatives of TarP were evaluated for their ability to support the recruitment of EGFP-Cdc42 to the plasma membrane. By enumerating the incidence of recruitment, we found that those constructs that retained recruitment of FAK and actin also were able to recruit Cdc42. Fig. 9B showed that TarP1–714 was able to support Cdc42 recruitment, but progressive deletion (TarP1–639) significantly reduced recruitment incidence to the level of the negative control TirM (3-fold difference versus TirM; p < 0.005). Furthermore, TarP-LD was sufficient to recruit Cdc42 (3-fold difference versus TirM; p < 0.001). Importantly, the conserved Leu residues were essential, with TarP-mutLD displaying a 3-fold decrease in EGFP-Cdc42 recruitment (Fig. 9B). Fig. 9C shows representative images of EGFP-Cdc42 recruitment by select TirM-TarP constructs.

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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Related in: MedlinePlus