The Chlamydiae are obligate intracellular pathogens that cause a range of acute and chronic diseases including trachoma, conjunctivitis, pelvic inflammatory disease and atypical pneumonias. In infected cells, Chlamydia efficiently acquires host-derived membrane lipids despite an apparent isolation of the bacterial - containing vacuole (""""""""inclusion"""""""") from classical endocytic and secretory membrane traffic. Because Chlamydiae are not amenable to genetic manipulation, little is known about the molecular basis for bacteria- mediated acquisition of eukaryotic lipids. ? ? We have determined that Chlamydia trachomatis synthesizes proteins that target eukaryotic lipid droplets (LD) - a lipid storage organelle. We have characterized the interaction between C. trachomatis and LDs and found that chlamydial infection led to the proliferation of a subclass of LDs on the surface of the inclusion. Furthermore, pharmacological disruption of LD biogenesis inhibited chlamydial replication. We hypothesize that Chlamydia co-opts the function of LDs to establish a growth permissive environment within infected cells. ? ? We propose to: 1) characterize the impact of chlamydial infection on LD functions by cell biological and proteomic approaches and to determine the role of individual LDs proteins in Chlamydia pathogenesis by RNA interference-mediated disruption of key LDs components, 2) identify the cellular targets of Chlamydia LD-tropic proteins through state-of-the art yeast genomics tools and affinity-based isolation of binding partners from mammalian expression cell lines. ? ? This is the first example of a bacterial pathogen targeting LDs, providing a unique opportunity to investigate a novel mechanism of pathogen-mediated eukaryotic organelle subversion. Our proposed approaches will define the cellular pathways affected by Chlamydia LD-tropic proteins, identity novel targets for therapeutic intervention and establish the role of these poorly characterized organelles in Chlamydia infection. ? ? ?
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