Limits...
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.

Show MeSH

Related in: MedlinePlus

Recruitment of GFP-actin after binding of 40 µm beads coated with α-CD4 antibody to cells expressing the fusion protein CD4-1xR and its mutant derivatives.a) The wild-type and mutant sequence of the second repeat of the TARP protein from the C. trachomatis serovar L2 is shown, from amino acid 174 to 222, the amino acid sequence of the Immunoreceptor Tyrosine-based Activator Motif (ITAM), and the surrounding amino acids of the tyrosine residue in the Tir protein essential for actin pedestal formation by EPEC to highlight the potential for this region of the TARP protein to be recognized by signaling molecules that are known to participate in actin cytoskeletal remodeling. Relevant tyrosine and substituted phenylalanine residues are in bold. Western blot of total lysates prepared from cells transfected with the various CD4-1xR expression constructs demonstrates equal levels of expression of the different derivatives of the CD4-1xR fusion protein and their respective reactivity to the 4G10 anti-phosphotyrosine antibody; b) Recruitment of GFP-actin upon aggregation of the plasma membrane-localized CD4-1xR fusion derivatives after incubation with beads coated with the monoclonal anti-CD4 antibody. Note the lack of GFP-actin recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. c) Localization of GFP-Rac at the sites of CD4-1xR aggregation by the anti-CD4 antibody-coated beads. Note the lack of GFP-Rac recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. In addition, membrane ruffles could be observed, which is typically found in cells that overexpress Rac. Asterisks indicate the position of the beads. Scale bars = 10 µm
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279300&req=5

ppat-1000014-g001: Recruitment of GFP-actin after binding of 40 µm beads coated with α-CD4 antibody to cells expressing the fusion protein CD4-1xR and its mutant derivatives.a) The wild-type and mutant sequence of the second repeat of the TARP protein from the C. trachomatis serovar L2 is shown, from amino acid 174 to 222, the amino acid sequence of the Immunoreceptor Tyrosine-based Activator Motif (ITAM), and the surrounding amino acids of the tyrosine residue in the Tir protein essential for actin pedestal formation by EPEC to highlight the potential for this region of the TARP protein to be recognized by signaling molecules that are known to participate in actin cytoskeletal remodeling. Relevant tyrosine and substituted phenylalanine residues are in bold. Western blot of total lysates prepared from cells transfected with the various CD4-1xR expression constructs demonstrates equal levels of expression of the different derivatives of the CD4-1xR fusion protein and their respective reactivity to the 4G10 anti-phosphotyrosine antibody; b) Recruitment of GFP-actin upon aggregation of the plasma membrane-localized CD4-1xR fusion derivatives after incubation with beads coated with the monoclonal anti-CD4 antibody. Note the lack of GFP-actin recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. c) Localization of GFP-Rac at the sites of CD4-1xR aggregation by the anti-CD4 antibody-coated beads. Note the lack of GFP-Rac recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. In addition, membrane ruffles could be observed, which is typically found in cells that overexpress Rac. Asterisks indicate the position of the beads. Scale bars = 10 µm

Mentions: Upon interaction of the EBs with epithelial cells, the TARP tyrosine residues that are phosphorylated are within the context of the phosphorylation sites for members of the Src-family of kinases and recognition sites of various src-homology 2 domain (SH2)-containing adaptor proteins (Figure 1a). The presence of these sequences raises the possibility that TARP may recruit signaling molecules that recruit and remodel actin. To directly test this hypothesis, a mammalian expression vector with an insert that encodes for a fusion protein containing the N-terminal extracellular domain of CD4 (amino acids 1–372) and one phosphodomain unit, with wild type or mutant sequences was synthesized, and co-transfected into Cos-7 cells along with either GFP-actin or GFP-Rac1. The second repeated (amino acids 174–222) unit has the sequence DAAADYEPISTTENIYESIDDSSTSDPENTSGGAAALNSLRGSSYSNYD, with the relevant tyrosines underlined (Figure 1a). These tyrosine residues were targeted because they are in the context of the recognition motifs for various Src kinase family and SH2 domain-containing adapter proteins [17]. Interestingly, these features of the tyrosines in the TARP phosphodomain are shared by the critical tyrosine in the Tir protein of enteropathogenic E. coli [18].


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 GFP-actin after binding of 40 µm beads coated with α-CD4 antibody to cells expressing the fusion protein CD4-1xR and its mutant derivatives.a) The wild-type and mutant sequence of the second repeat of the TARP protein from the C. trachomatis serovar L2 is shown, from amino acid 174 to 222, the amino acid sequence of the Immunoreceptor Tyrosine-based Activator Motif (ITAM), and the surrounding amino acids of the tyrosine residue in the Tir protein essential for actin pedestal formation by EPEC to highlight the potential for this region of the TARP protein to be recognized by signaling molecules that are known to participate in actin cytoskeletal remodeling. Relevant tyrosine and substituted phenylalanine residues are in bold. Western blot of total lysates prepared from cells transfected with the various CD4-1xR expression constructs demonstrates equal levels of expression of the different derivatives of the CD4-1xR fusion protein and their respective reactivity to the 4G10 anti-phosphotyrosine antibody; b) Recruitment of GFP-actin upon aggregation of the plasma membrane-localized CD4-1xR fusion derivatives after incubation with beads coated with the monoclonal anti-CD4 antibody. Note the lack of GFP-actin recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. c) Localization of GFP-Rac at the sites of CD4-1xR aggregation by the anti-CD4 antibody-coated beads. Note the lack of GFP-Rac recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. In addition, membrane ruffles could be observed, which is typically found in cells that overexpress Rac. Asterisks indicate the position of the beads. Scale bars = 10 µm
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000014-g001: Recruitment of GFP-actin after binding of 40 µm beads coated with α-CD4 antibody to cells expressing the fusion protein CD4-1xR and its mutant derivatives.a) The wild-type and mutant sequence of the second repeat of the TARP protein from the C. trachomatis serovar L2 is shown, from amino acid 174 to 222, the amino acid sequence of the Immunoreceptor Tyrosine-based Activator Motif (ITAM), and the surrounding amino acids of the tyrosine residue in the Tir protein essential for actin pedestal formation by EPEC to highlight the potential for this region of the TARP protein to be recognized by signaling molecules that are known to participate in actin cytoskeletal remodeling. Relevant tyrosine and substituted phenylalanine residues are in bold. Western blot of total lysates prepared from cells transfected with the various CD4-1xR expression constructs demonstrates equal levels of expression of the different derivatives of the CD4-1xR fusion protein and their respective reactivity to the 4G10 anti-phosphotyrosine antibody; b) Recruitment of GFP-actin upon aggregation of the plasma membrane-localized CD4-1xR fusion derivatives after incubation with beads coated with the monoclonal anti-CD4 antibody. Note the lack of GFP-actin recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. c) Localization of GFP-Rac at the sites of CD4-1xR aggregation by the anti-CD4 antibody-coated beads. Note the lack of GFP-Rac recruitment in the F1F2 mutant, indicating the requirement for the presence of at least one of the two tyrosine residues within the ITAM sequence. In addition, membrane ruffles could be observed, which is typically found in cells that overexpress Rac. Asterisks indicate the position of the beads. Scale bars = 10 µm
Mentions: Upon interaction of the EBs with epithelial cells, the TARP tyrosine residues that are phosphorylated are within the context of the phosphorylation sites for members of the Src-family of kinases and recognition sites of various src-homology 2 domain (SH2)-containing adaptor proteins (Figure 1a). The presence of these sequences raises the possibility that TARP may recruit signaling molecules that recruit and remodel actin. To directly test this hypothesis, a mammalian expression vector with an insert that encodes for a fusion protein containing the N-terminal extracellular domain of CD4 (amino acids 1–372) and one phosphodomain unit, with wild type or mutant sequences was synthesized, and co-transfected into Cos-7 cells along with either GFP-actin or GFP-Rac1. The second repeated (amino acids 174–222) unit has the sequence DAAADYEPISTTENIYESIDDSSTSDPENTSGGAAALNSLRGSSYSNYD, with the relevant tyrosines underlined (Figure 1a). These tyrosine residues were targeted because they are in the context of the recognition motifs for various Src kinase family and SH2 domain-containing adapter proteins [17]. Interestingly, these features of the tyrosines in the TARP phosphodomain are shared by the critical tyrosine in the Tir protein of enteropathogenic E. coli [18].

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