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Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

Lane BJ, Mutchler C, Al Khodor S, Grieshaber SS, Carabeo RA - PLoS Pathog. (2008)

Bottom Line: The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment.The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate.Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Louisville Medical School, Louisville, Kentucky, USA.

ABSTRACT
Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

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The p85 subunit of phosphatidylinositol 3-kinase interacts with the phosphodomain of TARP.a) Binding of CMTPX-labeled C. trachomatis L2 EBs to cells expressing the PI 3,4,5-P3 probe GFP-BTK-PH induces the localized synthesis of the lipid, and thus recruitment of the fluorescent probe. The images were obtained by live cell microscopy at 5 sec intervals. Treatment with the PI3-kinase inhibitor, wortmannin (100 nM) abolished PI 3,4,5-P3 synthesis, demonstrating the specificity of the GFP-BTK-PH probe. B) Oligopeptide pulldown using HeLa cell lysates demonstrate the interaction of the SH2 domain-containing p85 subunit of PI3-kinase in a phosphorylation-dependent manner, showing preference for the pY1 oligopeptide.
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ppat-1000014-g003: The p85 subunit of phosphatidylinositol 3-kinase interacts with the phosphodomain of TARP.a) Binding of CMTPX-labeled C. trachomatis L2 EBs to cells expressing the PI 3,4,5-P3 probe GFP-BTK-PH induces the localized synthesis of the lipid, and thus recruitment of the fluorescent probe. The images were obtained by live cell microscopy at 5 sec intervals. Treatment with the PI3-kinase inhibitor, wortmannin (100 nM) abolished PI 3,4,5-P3 synthesis, demonstrating the specificity of the GFP-BTK-PH probe. B) Oligopeptide pulldown using HeLa cell lysates demonstrate the interaction of the SH2 domain-containing p85 subunit of PI3-kinase in a phosphorylation-dependent manner, showing preference for the pY1 oligopeptide.

Mentions: Because the presence of PI 3,4,5-P3 appears to be necessary for optimum GEF activity of Vav2 towards Rac, the localized synthesis of this phospholipid at the site of chlamydial entry was investigated, using the probe BTK-PH-GFP, where the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (BTK) was fused to GFP. This domain has been demonstrated to be specific for PI 3,4,5-P3 [23]–[27]. Cells expressing this probe were infected by CMTPX-labeled C. trachomatis serovar L2, and monitored using live microscopy (Figure S2). As shown in Figure 3, localized bursts of EGFP-BTK-PH recruitment could be observed. Both the recruitment and disappearance of the fluorescent probe were rapid and transient. To test if the recruitment of BTK-PH-GFP was due to PI 3,4,5-P3 synthesis, transfected cells pre-treated with 100 nM wortmannin were monitored by live cell microscopy. As shown in Figure 3a and Figure S3, BTK-PH-GFP recruitment at the site of entry was not observed. Thus, BTK-PH-GFP localization at the site of entry was likely associated with PI 3,4,5-P3 synthesis. This localized PI 3,4,5-P3 synthesis would be expected to participate in the activation of Rac1 by the Vav2 GEF. That not all EBs localized with the BTK-PH-EGFP reporter could be attributed to the presence of a relatively large number of non-infectious EB particles, which is common in purified EB preparations, or that some simply do not utilize the PI3-kinase pathway of chlamydia entry.


Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

Lane BJ, Mutchler C, Al Khodor S, Grieshaber SS, Carabeo RA - PLoS Pathog. (2008)

The p85 subunit of phosphatidylinositol 3-kinase interacts with the phosphodomain of TARP.a) Binding of CMTPX-labeled C. trachomatis L2 EBs to cells expressing the PI 3,4,5-P3 probe GFP-BTK-PH induces the localized synthesis of the lipid, and thus recruitment of the fluorescent probe. The images were obtained by live cell microscopy at 5 sec intervals. Treatment with the PI3-kinase inhibitor, wortmannin (100 nM) abolished PI 3,4,5-P3 synthesis, demonstrating the specificity of the GFP-BTK-PH probe. B) Oligopeptide pulldown using HeLa cell lysates demonstrate the interaction of the SH2 domain-containing p85 subunit of PI3-kinase in a phosphorylation-dependent manner, showing preference for the pY1 oligopeptide.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279300&req=5

ppat-1000014-g003: The p85 subunit of phosphatidylinositol 3-kinase interacts with the phosphodomain of TARP.a) Binding of CMTPX-labeled C. trachomatis L2 EBs to cells expressing the PI 3,4,5-P3 probe GFP-BTK-PH induces the localized synthesis of the lipid, and thus recruitment of the fluorescent probe. The images were obtained by live cell microscopy at 5 sec intervals. Treatment with the PI3-kinase inhibitor, wortmannin (100 nM) abolished PI 3,4,5-P3 synthesis, demonstrating the specificity of the GFP-BTK-PH probe. B) Oligopeptide pulldown using HeLa cell lysates demonstrate the interaction of the SH2 domain-containing p85 subunit of PI3-kinase in a phosphorylation-dependent manner, showing preference for the pY1 oligopeptide.
Mentions: Because the presence of PI 3,4,5-P3 appears to be necessary for optimum GEF activity of Vav2 towards Rac, the localized synthesis of this phospholipid at the site of chlamydial entry was investigated, using the probe BTK-PH-GFP, where the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (BTK) was fused to GFP. This domain has been demonstrated to be specific for PI 3,4,5-P3 [23]–[27]. Cells expressing this probe were infected by CMTPX-labeled C. trachomatis serovar L2, and monitored using live microscopy (Figure S2). As shown in Figure 3, localized bursts of EGFP-BTK-PH recruitment could be observed. Both the recruitment and disappearance of the fluorescent probe were rapid and transient. To test if the recruitment of BTK-PH-GFP was due to PI 3,4,5-P3 synthesis, transfected cells pre-treated with 100 nM wortmannin were monitored by live cell microscopy. As shown in Figure 3a and Figure S3, BTK-PH-GFP recruitment at the site of entry was not observed. Thus, BTK-PH-GFP localization at the site of entry was likely associated with PI 3,4,5-P3 synthesis. This localized PI 3,4,5-P3 synthesis would be expected to participate in the activation of Rac1 by the Vav2 GEF. That not all EBs localized with the BTK-PH-EGFP reporter could be attributed to the presence of a relatively large number of non-infectious EB particles, which is common in purified EB preparations, or that some simply do not utilize the PI3-kinase pathway of chlamydia entry.

Bottom Line: The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment.The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate.Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Louisville Medical School, Louisville, Kentucky, USA.

ABSTRACT
Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

Show MeSH
Related in: MedlinePlus