The obligate intracellular bacterial pathogen Chlamydia trachomatis is responsible for a range of ocular and genital infections of significant clinical importance. The pathogen multiplies in epithelial cells within a membrane-bound vacuole named the inclusion. Though it is a crucial step in their pathogenesis, the mechanisms used by Chlamydiae to transport nutrients into the inclusion are poorly characterized. Studies with pharmacological inhibitors indicate that the acquisition of host cell-derived lipids into the inclusion is essential for bacterial replication. In eukaryotic cells, lipid droplets (LD), organelles consisting in a core of neutral lipids, constitute the main store of lipids. In Chlamydia-infected cells, LD accumulate at the periphery of the inclusion in a process mediated by chlamydial proteins, strongly suggesting that this pathogen targets LD to obtain a source of lipids. Recent findings made by Dr. Valdivia’s group, indicate that Chlamydia induces the translocation of intact cytoplasmic LD into the inclusion lumen. This represents the first example of mammalian LD functions being co�opted by a bacterial pathogen and points out to previously unrecognized host-pathogen interactions. In this project, I propose to perform a molecular characterization of this completely novel pathogenic mechanism. As a first step, I will use mass spectrometry technology for the qualitative and quantitative characterization of LD-associated proteins from both, infected and uninfected cells, in order to identify proteins that are differentially recruited to LD during infection. This approach will also allow the identification of bacterial factors potentially involved in this process. I will then use siRNA technology and classical cell biology techniques to determine the role of: a) selected candidates identified in the proteomic screen and b) cytoskeletal motors and host cell proteins involved in vesicular transport (eg. Rab and Rab effectors) in regulating LD-translocation into the inclusion lumen. The completion of this project will provide new insight into the molecular basis of how Chlamydia manipulates the host cell to subvert LD and the cell biology of these understudied organelles.