Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia Trachomatis Tarp During Infectivity and Reveals a Specific Role for the C Terminus During Cellular Invasion

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes , we speculated as to what degree Tarp function contributes to 's ability to parasitize mammalian host cells. To address this question, we generated a complete deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in with wild-type or mutant alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type entry into HeLa cells resulted in host cell shape changes, whereas the mutant did not. Finally, using a novel complementation approach, lacking demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide virulence factor.