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). secreted effector protein called Tarp that is translocated upon contact with host cells and independently nucleates actin filament formation. Tarp from a lymphogranuloma venereum (LGV) strain consists of a tyrosine-rich repeat domain, a proline-rich domain required for oligomerization, and a single actin binding domain. Oligomerization is required to bring multiple actin monomers together to initiate actin filament formation by a mechanism distinct from host actin nucleators. Here we have PPARG2 examined Tarp from several other strains of chlamydiae and find that certain of these contain up to four actin binding domains. Tarp fragments bearing multiple actin binding domains nucleate actin in assays even in the absence of the oligomerization domain. This suggests that Tarp from different chlamydial species may utilize hybrid mechanisms to induce actin nucleation. Determination of virulence determinants in chlamydiae is challenging due to the lack of tractable genetic systems. The direct introduction of anti-Tarp actin binding domain antibodies into the cytosol of host cells inhibited entry and thus demonstrates an essential role for Tarp in chlamydial pathogenesis. Introduction The obligate intracellular gram negative bacterium, directly induces actin polymerization via the type III secreted effector, Tarp . elementary bodies harbor presynthesized Tarp effector protein which is tyrosine phosphorylated upon translocation to the cytosol of the host cell and has been implicated in the recruitment of actin observed at the site HCV-IN-3 of chlamydial attachment L2 Tarp harbors at least three functionally distinct domains; an N-terminal tyrosine-rich repeat domain of unknown function, a proline rich domain required for Tarp oligomerization and a single Wiskott-Aldrich syndrome protein (WASP)-Homology-2 (WH2) G-actin binding domain , . The proline rich domain and actin binding domain are harbored within the minimum Tarp peptide required for Tarp mediated actin nucleation. Oligomerization mediated by the proline-rich domain presumably brings multiple actin monomers into apposition to nucleate actin filament formation . Sequence analysis of all known Tarp orthologs indicate that HCV-IN-3 the proline rich domain and actin binding alpha helix are conserved although the tyrosine-rich repeat domain is absent from and various serovars all harbor at least one and up to four functional actin binding domains and that purified recombinant Tarps from all chlamydial species were capable of nucleating actin filament formation L2 Tarp Three distinct domains of the Tarp effector have been described. These include: the tyrosine rich repeat region (amino acids 125C424), a proline rich domain required for Tarp multimerization (amino acids 625C650), and an actin binding domain (amino acids 748C758) , , , . Only the proline rich domain and actin binding domain appear to be conserved among chlamydial species. To determine the significance of the actin binding domain, an anti-peptide antibody to amino acids 746C760 encompassing that region was produced in rabbits and antigen-affinity purified HCV-IN-3 to generate the anti actin binding domain (ABD) antibody. This antibody demonstrated affinity to the actin binding domain as confirmed by immunoblots using a series of GST-L2-Tarp fusion proteins (Figure 1A). Western blot analysis of chlamydia-infected host cells with the ABD antibody confirmed the antibody recognizes Tarp in the infected lysates but did not HCV-IN-3 detectably cross-react with any host proteins (Figure 1B). The affinity purified L2 Tarp ABD antibody also recognized an immunodominant protein in lysates generated from purified EBs of other chlamydial species including (Figure 1C). Open in a separate window 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.