Figure 1.
The Tarp actin binding domain (ABD) peptide antibody recognizes native Tarp of multiple serovars and species and does not recognize the ABD of the host cell WAVE2 protein.
A) Schematic of C. trachomatis GST-Tarp fusions used to examine the specificity of the peptide antibody directed toward the Tarp actin binding domain. Tarp amino acids and positions are indicated above each bar in the schematic. Purified GST fusions were immobilized to nitrocellulose and immunoblots were performed with Tarp actin binding domain (αABD) and Tarp (α Tarp) specific antibodies. B) The Tarp actin binding domain (α ABD) specific antisera recognizes only a single protein within chlamydia-infected host cells. Chlamydia-infected (+L2) and uninfected (−L2) host cells were suspended in protein sample buffer following a 30 min. infection. Proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (CB). Immunoblots were performed with Tarp (α Tarp) and Tarp actin binding domain (α ABD) specific antisera. C) The Tarp actin binding domain (α ABD) antibody recognizes a protein in lysates generated from purified C. trachomatis serovar L2 (LGV-434), C. caviae (GPIC), C. pneumoniae (Cpn), C. trachomatis serovar D (D-UW3), C. trachomatis serovar A (A HAR-13) and C. muridarum mouse pneumonitis biovar (MoPn) elementary bodies. Loading for SDS-PAGE was based upon equivalent numbers of EBs. C. pneumoniae Tarp was not readily visible on the original exposure but was easily visualized with longer exposures. D) The Tarp actin binding domain (α ABD) antibody recognizes non-reduced, non-denatured native protein immobilized to nitrocellulose. Immunoblots were performed of lysates generated from cells infected with C. trachomatis (HeLa +L2) and uninfected host cells (HeLa). Purified recombinant Tarp protein (C-domain Tarp) and solubilized lysates derived from elementary bodies (EBs) served as positive controls. Immunoblots to detect WAVE2 (α WAVE2) and actin (α actin) were performed as additional controls. E) The Tarp actin binding domain (α ABD) antibody immunoprecipitates Tarp from infected cells. Tarp was immunoprecipitated with α ABD from lysates generated from cells infected with C. trachomatis (HeLa +L2) and uninfected host cells (HeLa). Proteins were resolved by SDS-PAGE and immunoblotted with Tarp (α Tarp) and WAVE2 (α WAVE2) specific antibodies (arrowheads). The anti-Tarp polyclonal antibody recognizes an unknown antigen in the infected and uninfected HeLa cell lysates that is not immunoprecipitated by the α ABD antibody. The αABD antibody does not recognize this antigen in immunoblots (panels B, D, and F). Note that the IgG heavy chain is observed in both infected and uninfected lanes. F) Tarp and WAVE2 were immunoprecipitated from infected (+L2) and uninfected (−L2) HeLa cells, resolved by SDS-PAGE and immunoblotted with Tarp actin binding domain (α ABD) and WAVE2 (α WAVE2) specific antibodies (arrowheads). Molecular mass is in kilodaltons (kDa) for panels B, C, E & F.
Figure 2.
The Tarp actin binding domain (ABD) peptide antibody inhibits Tarp mediated actin polymerization in vitro and inhibits chlamydial entry in vivo.
A) Tarp mediated actin nucleation (Tarp) was inhibited by the addition of Tarp actin binding domain specific antisera (Tarp+ABD Ab). Purified Tarp, GST and antibodies were added to 1µM pyrene conjugated actin and actin polymerization was measured as arbitrary fluorescence intensity (Intensity a.u.) over time (Time seconds) following the addition of polymerization buffer at 300 seconds. An irrelevant antibody did not alter Tarp (Tarp+control Ab) or GST (GST+control Ab) mediated actin polymerization. GST (GST) and actin alone (actin alone) served as additional controls. B) Graphical representation of EB invasion of ABD antibody pre-loaded HeLa cells. HeLa cells were pre-loaded with ABD or nonspecific control antibodies (control Ab) using a cationic lipid mixture (Pro-Ject Protein Transfection Reagent) to deliver the antibodies to the host cytosol. Intrinsically fluorescent CMPTX labeled EBs were used in invasion assays. After allowing for 30 min invasion, extracellular EBs were counterstained by indirect immunofluorescence with a monoclonal antibody to C. trachomatis L2 MOMP and a goat anti mouse antibody conjugated to Alexa 488. The percent of EB invasion (% invasion) was determined for cells harboring purified ABD (ABD Ab), ABD preincubated with an excess of the peptide immunogen (ABD Ab+peptide) and irrelevant control antibody (control Ab). Additional controls included untreated host cells (No Ab). The results are from one experiment representative of three separate experiments.
Figure 3.
Tarp orthologs harbor multiple actin binding domains.
A) A schematic of the Tarp orthologs from C. trachomatis serovar L2 (L2), C. trachomatis serovar D (D), C. trachomatis serovar A (A), C. muridarum (MoPn), C. pneumoniae (Cpn), and C. caviae (GPIC) indicating the location of the putative actin binding domains (red boxes), a proline rich domain (blue boxes), and tyrosine rich phosphorylation domain (green boxes). B) ClustalW sequence alignment of the putative actin binding domains from Tarp orthologs in A. The sequence predicted to harbor the actin binding alpha helix is indicated by the open box. Identical amino acids within each alignment are in red. Similar residues are in blue. The consensus sequence shown is based on homology greater than 50%. The number indicates the amino acid residue of the amino terminus of the peptide shown. C) The Tarp orthologs associate with actin. GST-fusions of the Tarp orthologs described above harboring sequence similar to the C. trachomatis L2 (L2) actin binding domain were expressed and purified. Extracts from HeLa cells were incubated with GST or GST fusions to Tarp orthologs and specifically bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (CB). Samples identical to those shown in the Coomassie-stained gel were subject to immunoblotting with an actin (α actin) specific antibody. A GST fusion to the VCA domain of N-wasp (GST-VCA) served as a positive control for actin binding.
Figure 4.
C. caviae, C. pneumoniae and C. trachomatis Tarp orthologs promote actin polymerization.
A) Pyrene actin polymerization in the presence of GST-Tarp fusions. GST-Tarp fusions representing the C-domain of C. trachomatis L2 (L2), C. trachomatis serovar D (D) C. trachomatis A HAR13 (A) and C. muridarum (MoPn) were incubated with 1µM pyrene conjugated actin and actin polymerization was measured as arbitrary fluorescence intensity (Intensity a.u.) over time (Time seconds) following the addition of polymerization buffer at 300 seconds. GST and actin alone served as negative controls. B) Pyrene actin polymerization assays as A but with GST-Tarp fusions of the C-domain of C. caviae (GPIC), C. trachomatis L2 (L2), and negative controls GST, and actin alone. C) Pyrene actin polymerization assays as A but with GST-Tarp fusions of C. pneumoniae (CPn), C. trachomatis L2 (L2), and negative controls GST, and actin alone. D) Pyrene actin polymerization assays as A but with GST-Tarp fusions of Tarp orthologs lacking the proline rich domain. Note that all Tarp orthologs with greater than one actin binding domain increase initial rates of actin polymerization over the GST control. As previously shown [14], C. trachomatis L2 Tarp lacking the proline rich domain sequesters actin monomers to depress actin nucleation rates below control levels. The results are from one experiment representative of three separate experiments.
Figure 5.
The C. trachomatis serovar A Tarp ortholog employs a spire-like actin nucleation mechanism and does not require the L2 Tarp proline rich domain for actin nucleation.
C. trachomatis serovar A Tarp fragments harboring either the three functional actin binding domains (ABDs) alone or the actin binding domains and the proline rich domain (PRD) were digested to remove the GST moiety and analyzed by gel filtration and pyrene actin polymerization assays. A) C. trachomatis serovar A GST-Tarp fusion proteins were purified and digested with protease (+/− enz) to remove the GST moiety (* indicates GST is removed). Proteins were resolved by SDS/PAGE and visualized by Coomassie blue staining. B) Removal of the proline rich domain from C. trachomatis A Tarp inhibits oligomerization. Gel filtration of proteins shown in panel A. Protein fractions were collected in 2-min intervals from gel filtration columns and immobilized to a nitrocellulose membrane by vacuum filtration. Membranes were subjected to immunoblotting with a Tarp specific antibody. Protein standards are indicated above the dot-blot with respective molecular weight and peak elution times. C) Oligomerization of C. trachomatis A Tarp is not required for actin nucleation. Purified Tarp (A) with and without proline rich domain increased actin polymerization compared to GST and actin alone controls in pyrene actin polymerization assays. The results are from one experiment representative of three separate experiments.