The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated obligate pathogen that infects epithelial surfaces of the urogenital tract, leading to severe sequela such as pelvic inflammatory disease, and infertility. During the initial stages of infection Chlamydia likely delivers between 10-15 separate Type III secreted (T3S) ?effector? proteins into the target host cell. We hypothesize that these Chlamydia effector proteins work co-operatively to temporally reprogram signaling pathways and cytoskeletal functions to promote cell invasion and establish a nascent pathogenic vacuole (?inclusion?) In our first aim, we propose to apply emerging genetic and molecular genetic tools in Chlamydia to define the role of early effectors play in invasion, inclusion maturation and in vivo infections. We also propose to perform an epistatic analysis of combinations of effector mutants to group effector by functional groups and to prioritize a detailed molecular characterization of effector that are central signaling nodes. In our second aim, we focus on the characterization of Tepp, an effector that plays important roles in reprograming signaling pathways, including those involved in innate immune responses. We propose to define the mechanism of activation of Class I phosphoinositide 3 kinases (PI3K) at nascent inclusions and the role these activities play in the regulation of membrane dynamics and activation of Type I interferon responses. In parallel, we will identify the molecular players that lead to the Tepp- mediated activation and modification of Eps8, a regulator of Rac1 activity and cell-cell junctions, by applying ?proximity proteomics? approaches. Overall, our research plan seeks to perform both a systems levels assessment of the function of early effectors and a detailed molecular characterization of mechanism of action for selected effectors. The overall goal is to define the molecular basis of how specific effector(s) function, identify how signaling pathways are re-programmed and how they all ultimately contribute to Chlamydia survival in host tissues and the induction of pathology.
(Public Health Relevance Statement) Chlamydia trachomatis is an obligate intracellular pathogen responsible for significant morbidity worldwide and a major contributor to impaired reproductive health in women. The molecular mechanisms of C. trachomatis invasion of mammalian cell are poorly understood because until recently this bacterium was not amenable to routine molecular genetic experimentation. This proposal uses state-of-the-art genetic and proteomic approaches to 1) define key steps in early invasion by the pathogen, and 2) characterize the function of key virulence factor of Chlamydia. These studies will ultimately contribute to the identification of novel potential targets for therapeutic intervention.
