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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

Characterization of the fraction precipitated by the various biotinylated oligopeptide derivatives.a) Western blot of the precipitated fractions to demonstrate the presence of various host proteins that participate in guanine nucleotide exchange of the Rac GTPase. HeLa cell lysates were incubated with the different phosphorylation derivatives of an oligopeptide that spans one unit of the repeated domain. Note the differences in the proteins pulled down by the different oligopeptides. b) The addition of the PI 3,4,5-P3 analog to the lysate prior to pulldown enhanced the interaction of Vav2 with Rac1. The intramolecular interaction of Vav2 blocks the binding domain of Rac1, which the lipid analog unmasked, and thus allowed for binding of Rac with Vav2. c) The interaction of the Sos1/Abi1/Eps8 complex with TARP requires the Abi1 protein. Lysates depleted of the various components of this complex were subjected to co-precipitation with the pY1 oligopeptide to determine the nature of its interaction with TARP. Abi1 was required for the coprecipitation of Sos1 and Eps8 with the pY1 oligopeptide, while Sos1 or Eps8 depletion did not affect the interaction of Abi1 with pY1, but rather partially inhibited their respective interactions with pY1. The red borders indicate the blots demonstrating efficiency of siRNA depletion, while the lack of the border signified the coprecipitated levels of the proteins.
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ppat-1000014-g002: Characterization of the fraction precipitated by the various biotinylated oligopeptide derivatives.a) Western blot of the precipitated fractions to demonstrate the presence of various host proteins that participate in guanine nucleotide exchange of the Rac GTPase. HeLa cell lysates were incubated with the different phosphorylation derivatives of an oligopeptide that spans one unit of the repeated domain. Note the differences in the proteins pulled down by the different oligopeptides. b) The addition of the PI 3,4,5-P3 analog to the lysate prior to pulldown enhanced the interaction of Vav2 with Rac1. The intramolecular interaction of Vav2 blocks the binding domain of Rac1, which the lipid analog unmasked, and thus allowed for binding of Rac with Vav2. c) The interaction of the Sos1/Abi1/Eps8 complex with TARP requires the Abi1 protein. Lysates depleted of the various components of this complex were subjected to co-precipitation with the pY1 oligopeptide to determine the nature of its interaction with TARP. Abi1 was required for the coprecipitation of Sos1 and Eps8 with the pY1 oligopeptide, while Sos1 or Eps8 depletion did not affect the interaction of Abi1 with pY1, but rather partially inhibited their respective interactions with pY1. The red borders indicate the blots demonstrating efficiency of siRNA depletion, while the lack of the border signified the coprecipitated levels of the proteins.

Mentions: Because one copy of the repeated unit is apparently sufficient to recruit actin and Rac1 in our cell culture assay, and the dependence of these activities on the phosphorylation of Tyr179 and Tyr189, it is hypothesized that the domain may act as a signaling platform to which host signaling molecules are recruited. To directly test this possibility, biotinylated oligopeptides with the sequence DAAADYEPISTTENIYESIDDSSTSDPENTSGGAAALNSLRGSSYSNYD were custom synthesized either as non-phosphorylated tyrosines (WT), individually phosphorylated tyrosines (pY1 and pY2), or in which phenylalanines have been substituted for the tyrosines (F1F2). The biotinylated oligopeptides were incubated with lysates from HeLa cells and the presence of molecules known to participate with Rac1 in signal transduction pathways, specifically the Rac guanine nucleotide exchange factors Vav2 and Sos1 were monitored by Western blotting. The specificity of Sos1 towards Rac1 is conferred by the Abi1 and Eps8 proteins [19]. Sos1 exclusively bound to the phosphorylated pY1 peptide, as shown in Figure 2a, while Abi1 bound equally well to pY1 and pY2 oligopeptides. Vav2 bound equally well to pY1 and pY2. Pulldown samples from the WT and F1F2 oligopeptides showed background or undetectable levels of the all the proteins monitored, indicative of the requirement for phosphorylated tyrosine residues.


Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

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

Characterization of the fraction precipitated by the various biotinylated oligopeptide derivatives.a) Western blot of the precipitated fractions to demonstrate the presence of various host proteins that participate in guanine nucleotide exchange of the Rac GTPase. HeLa cell lysates were incubated with the different phosphorylation derivatives of an oligopeptide that spans one unit of the repeated domain. Note the differences in the proteins pulled down by the different oligopeptides. b) The addition of the PI 3,4,5-P3 analog to the lysate prior to pulldown enhanced the interaction of Vav2 with Rac1. The intramolecular interaction of Vav2 blocks the binding domain of Rac1, which the lipid analog unmasked, and thus allowed for binding of Rac with Vav2. c) The interaction of the Sos1/Abi1/Eps8 complex with TARP requires the Abi1 protein. Lysates depleted of the various components of this complex were subjected to co-precipitation with the pY1 oligopeptide to determine the nature of its interaction with TARP. Abi1 was required for the coprecipitation of Sos1 and Eps8 with the pY1 oligopeptide, while Sos1 or Eps8 depletion did not affect the interaction of Abi1 with pY1, but rather partially inhibited their respective interactions with pY1. The red borders indicate the blots demonstrating efficiency of siRNA depletion, while the lack of the border signified the coprecipitated levels of the proteins.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000014-g002: Characterization of the fraction precipitated by the various biotinylated oligopeptide derivatives.a) Western blot of the precipitated fractions to demonstrate the presence of various host proteins that participate in guanine nucleotide exchange of the Rac GTPase. HeLa cell lysates were incubated with the different phosphorylation derivatives of an oligopeptide that spans one unit of the repeated domain. Note the differences in the proteins pulled down by the different oligopeptides. b) The addition of the PI 3,4,5-P3 analog to the lysate prior to pulldown enhanced the interaction of Vav2 with Rac1. The intramolecular interaction of Vav2 blocks the binding domain of Rac1, which the lipid analog unmasked, and thus allowed for binding of Rac with Vav2. c) The interaction of the Sos1/Abi1/Eps8 complex with TARP requires the Abi1 protein. Lysates depleted of the various components of this complex were subjected to co-precipitation with the pY1 oligopeptide to determine the nature of its interaction with TARP. Abi1 was required for the coprecipitation of Sos1 and Eps8 with the pY1 oligopeptide, while Sos1 or Eps8 depletion did not affect the interaction of Abi1 with pY1, but rather partially inhibited their respective interactions with pY1. The red borders indicate the blots demonstrating efficiency of siRNA depletion, while the lack of the border signified the coprecipitated levels of the proteins.
Mentions: Because one copy of the repeated unit is apparently sufficient to recruit actin and Rac1 in our cell culture assay, and the dependence of these activities on the phosphorylation of Tyr179 and Tyr189, it is hypothesized that the domain may act as a signaling platform to which host signaling molecules are recruited. To directly test this possibility, biotinylated oligopeptides with the sequence DAAADYEPISTTENIYESIDDSSTSDPENTSGGAAALNSLRGSSYSNYD were custom synthesized either as non-phosphorylated tyrosines (WT), individually phosphorylated tyrosines (pY1 and pY2), or in which phenylalanines have been substituted for the tyrosines (F1F2). The biotinylated oligopeptides were incubated with lysates from HeLa cells and the presence of molecules known to participate with Rac1 in signal transduction pathways, specifically the Rac guanine nucleotide exchange factors Vav2 and Sos1 were monitored by Western blotting. The specificity of Sos1 towards Rac1 is conferred by the Abi1 and Eps8 proteins [19]. Sos1 exclusively bound to the phosphorylated pY1 peptide, as shown in Figure 2a, while Abi1 bound equally well to pY1 and pY2 oligopeptides. Vav2 bound equally well to pY1 and pY2. Pulldown samples from the WT and F1F2 oligopeptides showed background or undetectable levels of the all the proteins monitored, indicative of the requirement for phosphorylated tyrosine residues.

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