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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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Focal adhesion components associate with TarP and its conserved LD-like motif.A, ClustalW sequence alignment showing the putative actin binding domains (ABD; open box) from Tarp orthologs with the conserved residues of the LD motif highlighted in yellow. The red asterisk indicates the recently identified F-actin binding domains (FAB1). The consensus sequence of the LD domain within TarP was aligned with the paxillin LD domain to highlight homology. Species analyzed include C. caviae (GPIC), Chlamydophila abortus (CAb), Chlamydophila felis (CFe), Chlamydia muridarum (MoPn), C. trachomatis serovars A, L2, and D. B, the TarP-LD-like motif was ectopically expressed in Cos7 cells for either 24 or 8 h and tested for the ability to co-recruit pY397-FAK and actin (white arrowheads). When transient expression was limited to 8 h rather than 24 h, the TarP-LD specifically localized to FAK-rich focal adhesion structures and to various points along actin stress fibers. Large arrowheads in the 24-h panel indicate the co-recruitment of pY397-FAK and actin to TarP-LD aggregates, whereas in the 8-h panel, they indicate the localization of TarP-LD to focal adhesion structures. Small arrowheads indicate the localization of TarP-LD along the F-actin. Scale bars, 10 μm.
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Figure 2: Focal adhesion components associate with TarP and its conserved LD-like motif.A, ClustalW sequence alignment showing the putative actin binding domains (ABD; open box) from Tarp orthologs with the conserved residues of the LD motif highlighted in yellow. The red asterisk indicates the recently identified F-actin binding domains (FAB1). The consensus sequence of the LD domain within TarP was aligned with the paxillin LD domain to highlight homology. Species analyzed include C. caviae (GPIC), Chlamydophila abortus (CAb), Chlamydophila felis (CFe), Chlamydia muridarum (MoPn), C. trachomatis serovars A, L2, and D. B, the TarP-LD-like motif was ectopically expressed in Cos7 cells for either 24 or 8 h and tested for the ability to co-recruit pY397-FAK and actin (white arrowheads). When transient expression was limited to 8 h rather than 24 h, the TarP-LD specifically localized to FAK-rich focal adhesion structures and to various points along actin stress fibers. Large arrowheads in the 24-h panel indicate the co-recruitment of pY397-FAK and actin to TarP-LD aggregates, whereas in the 8-h panel, they indicate the localization of TarP-LD to focal adhesion structures. Small arrowheads indicate the localization of TarP-LD along the F-actin. Scale bars, 10 μm.

Mentions: The interaction of FAK with a variety of binding partners either depends on phosphorylation (on tyrosine, serine, and/or threonine) or on signaling domains (FERM and FAT) (45). Amino acid sequence analysis of TarP orthologues from various chlamydial species for mammalian-like signaling motifs identified a domain (LD) similar to paxillin and thus potentially could be recognized by the FAT domain of FAK (Fig. 2A). These highly conserved LD-like motifs overlapped a region originally characterized as non-functional globular actin binding domain. In addition to the amino acid sequence conservation, the positions along the TarP orthologs were also conserved, with only the most C-terminal of the actin binding domains harboring the LD-like motifs. Thus, there appears to be both sequence and position conservation among the TarP-LD motifs found in various TarP orthologues. The most distinct feature of the TarP-LD sequences (LEXLLPXLRAHL) was the conserved leucine residues, which in paxillin LD2 are critical for FAK binding. The putative signaling function would be in contrast to the F-actin binding activity previously assigned to this region of TarP (18). Therefore, it was necessary to address this potential discrepancy. Using a similar approach used by the Jiwani et al. (18), ectopically expressed TarP-LD colocalized with F-actin as previously reported. However, when the duration of expression was limited to 8 h, colocalization was predominantly at the tips of stress fibers, which coincidentally were also positive for pY397-FAK (Fig. 2B). Overall, the data raised the possibility that the FAB1 motif may have an alternate function, which is to mediate TarP/FAK interaction at the plasma membrane during invasion.


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)

Focal adhesion components associate with TarP and its conserved LD-like motif.A, ClustalW sequence alignment showing the putative actin binding domains (ABD; open box) from Tarp orthologs with the conserved residues of the LD motif highlighted in yellow. The red asterisk indicates the recently identified F-actin binding domains (FAB1). The consensus sequence of the LD domain within TarP was aligned with the paxillin LD domain to highlight homology. Species analyzed include C. caviae (GPIC), Chlamydophila abortus (CAb), Chlamydophila felis (CFe), Chlamydia muridarum (MoPn), C. trachomatis serovars A, L2, and D. B, the TarP-LD-like motif was ectopically expressed in Cos7 cells for either 24 or 8 h and tested for the ability to co-recruit pY397-FAK and actin (white arrowheads). When transient expression was limited to 8 h rather than 24 h, the TarP-LD specifically localized to FAK-rich focal adhesion structures and to various points along actin stress fibers. Large arrowheads in the 24-h panel indicate the co-recruitment of pY397-FAK and actin to TarP-LD aggregates, whereas in the 8-h panel, they indicate the localization of TarP-LD to focal adhesion structures. Small arrowheads indicate the localization of TarP-LD along the F-actin. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Focal adhesion components associate with TarP and its conserved LD-like motif.A, ClustalW sequence alignment showing the putative actin binding domains (ABD; open box) from Tarp orthologs with the conserved residues of the LD motif highlighted in yellow. The red asterisk indicates the recently identified F-actin binding domains (FAB1). The consensus sequence of the LD domain within TarP was aligned with the paxillin LD domain to highlight homology. Species analyzed include C. caviae (GPIC), Chlamydophila abortus (CAb), Chlamydophila felis (CFe), Chlamydia muridarum (MoPn), C. trachomatis serovars A, L2, and D. B, the TarP-LD-like motif was ectopically expressed in Cos7 cells for either 24 or 8 h and tested for the ability to co-recruit pY397-FAK and actin (white arrowheads). When transient expression was limited to 8 h rather than 24 h, the TarP-LD specifically localized to FAK-rich focal adhesion structures and to various points along actin stress fibers. Large arrowheads in the 24-h panel indicate the co-recruitment of pY397-FAK and actin to TarP-LD aggregates, whereas in the 8-h panel, they indicate the localization of TarP-LD to focal adhesion structures. Small arrowheads indicate the localization of TarP-LD along the F-actin. Scale bars, 10 μm.
Mentions: The interaction of FAK with a variety of binding partners either depends on phosphorylation (on tyrosine, serine, and/or threonine) or on signaling domains (FERM and FAT) (45). Amino acid sequence analysis of TarP orthologues from various chlamydial species for mammalian-like signaling motifs identified a domain (LD) similar to paxillin and thus potentially could be recognized by the FAT domain of FAK (Fig. 2A). These highly conserved LD-like motifs overlapped a region originally characterized as non-functional globular actin binding domain. In addition to the amino acid sequence conservation, the positions along the TarP orthologs were also conserved, with only the most C-terminal of the actin binding domains harboring the LD-like motifs. Thus, there appears to be both sequence and position conservation among the TarP-LD motifs found in various TarP orthologues. The most distinct feature of the TarP-LD sequences (LEXLLPXLRAHL) was the conserved leucine residues, which in paxillin LD2 are critical for FAK binding. The putative signaling function would be in contrast to the F-actin binding activity previously assigned to this region of TarP (18). Therefore, it was necessary to address this potential discrepancy. Using a similar approach used by the Jiwani et al. (18), ectopically expressed TarP-LD colocalized with F-actin as previously reported. However, when the duration of expression was limited to 8 h, colocalization was predominantly at the tips of stress fibers, which coincidentally were also positive for pY397-FAK (Fig. 2B). Overall, the data raised the possibility that the FAB1 motif may have an alternate function, which is to mediate TarP/FAK interaction at the plasma membrane during invasion.

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