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.