The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion

Abstract

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells. Chlamydiae are bacterial obligate intracellular pathogens responsible for multiple human and veterinary diseases. The induction of cytoskeletal rearrangements to promote chlamydial internalization is partially mediated by a type III secreted effector protein called Tarp that is translocated upon contact with host cells and independently nucleates actin filament formation. Tarp from a C. trachomatis 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 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 in vitro 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.