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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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Related in: MedlinePlus

Recruitment of various host signaling molecules to the sites of entry of C. trachomatis L2 EBs.HeLa cells infected for 10 min were fixed and prepared for immunofluorescent staining using antibodies specific for Sos1, Abi1, Eps8, and Vav2. White arrowheads indicate protein puncta colocalizing with EBs. Bordered regions indicate areas enlarged in inset images. Scale bars = 10 µm.
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ppat-1000014-g004: Recruitment of various host signaling molecules to the sites of entry of C. trachomatis L2 EBs.HeLa cells infected for 10 min were fixed and prepared for immunofluorescent staining using antibodies specific for Sos1, Abi1, Eps8, and Vav2. White arrowheads indicate protein puncta colocalizing with EBs. Bordered regions indicate areas enlarged in inset images. Scale bars = 10 µm.

Mentions: Phalloidin and 4G10 antibodies also colocalize at the sites of entry, where TARP molecules are predicted to be translocated across the host plasma membrane [9],[14]. Interaction of Sos1, Eps8, Abi1, and Vav2 with TARP would result in the localization of these molecules at the sites of chlamydial entry. Indeed, colocalizations of Sos1, Eps8, Abi1, and Vav2 with invading EBs were observed by antibody staining and indirect immunofluorescence (Figure 4). All four proteins were present as distinct puncta. We observed that 30% of EBs colocalized with Sos1, 41% with Abi1, 30% with Eps8, and 24% with Vav2. The significance of these values is unclear as they could be skewed by the quality of EB preparations and the transient nature of the localization of the signaling molecules. This transient localization of the signaling molecules to the sites of entry may have prevented the visualization of some of these recruitment events in fixed cells. Another possibility is the utilization of alternate mechanisms for some of the invading EBs.


Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

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

Recruitment of various host signaling molecules to the sites of entry of C. trachomatis L2 EBs.HeLa cells infected for 10 min were fixed and prepared for immunofluorescent staining using antibodies specific for Sos1, Abi1, Eps8, and Vav2. White arrowheads indicate protein puncta colocalizing with EBs. Bordered regions indicate areas enlarged in inset images. Scale bars = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000014-g004: Recruitment of various host signaling molecules to the sites of entry of C. trachomatis L2 EBs.HeLa cells infected for 10 min were fixed and prepared for immunofluorescent staining using antibodies specific for Sos1, Abi1, Eps8, and Vav2. White arrowheads indicate protein puncta colocalizing with EBs. Bordered regions indicate areas enlarged in inset images. Scale bars = 10 µm.
Mentions: Phalloidin and 4G10 antibodies also colocalize at the sites of entry, where TARP molecules are predicted to be translocated across the host plasma membrane [9],[14]. Interaction of Sos1, Eps8, Abi1, and Vav2 with TARP would result in the localization of these molecules at the sites of chlamydial entry. Indeed, colocalizations of Sos1, Eps8, Abi1, and Vav2 with invading EBs were observed by antibody staining and indirect immunofluorescence (Figure 4). All four proteins were present as distinct puncta. We observed that 30% of EBs colocalized with Sos1, 41% with Abi1, 30% with Eps8, and 24% with Vav2. The significance of these values is unclear as they could be skewed by the quality of EB preparations and the transient nature of the localization of the signaling molecules. This transient localization of the signaling molecules to the sites of entry may have prevented the visualization of some of these recruitment events in fixed cells. Another possibility is the utilization of alternate mechanisms for some of the invading EBs.

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