The obligate intracellular bacterium Chlamydia trachomatis causes substantial morbidity in the US and world wide. An inability to manipulate the chlamydial genome has consequently produced a dearth of bona fide virulence factors. Hence, pathogenic mechanisms of chlamydial infections are poorly described and require further examination. A C. trachomatis effector called Tarp, for translocated actin recruiting protein is a candidate virulence factor. Tarp is tyrosine phosphorylated by a host cell kinase and is associated with actin recruitment during Chlamydiae entry. Tarp protein domains have been identified and include: i) an actin binding and nucleating domain, ii) a proline rich oligomerization domain and iii) a phosphorylation domain. The function of the Tarp phosphorylation domain is currently unknown and the focus of the attached grant proposal. Src-homology domain 2 (SH2) containing proteins are candidate Tarp interacting proteins. The purpose of this grant proposal is to identify Tarp binding proteins (those with and without SH2 domains) by using assays to detect protein-protein interactions such as GST-fusion pull-down assays, immunoprecipitation, and two commercially available assays designed to target SH2 domain containing proteins. And secondly, to assess any requirements for identified Tarp binding proteins in Chlamydiae entry and development. Recently, PIS kinase was implicated in association with phosphorylated Tarp. Modification of lipids by PI3K ultimately regulates a variety of cellular processes. We predict that Tarp recruitment of PI3K plays a vital role in the establishment of chlamydial infection and intend to examine a requirement for this association with chemical inhibits and interfering RNAs. Currently, a chlamydial vaccine is not available. Elucidation of Tarp interactions with the host is crucial for mapping signaling pathways used by C. trachomatis to establish residence within infected cells. A better understanding of the mechanisms employed by Chlamydiae to initiate a successful infection will result in the identification of novel therapeutic interventions. RELEVANCE: The sexually transmitted disease causing bacteria, Chlamydia trachomatis, infected more than 1 million people in the US alone last year. A recently identified Chlamydiae protein called Tarp is believed to play an important role in the initial stages of infection. The goal of this research proposal is to determine how the Tarp protein facilitates Chlamydiae to cause disease.