Chlamydia trachomatis is the leading cause of sexually transmitted diseases in this country and a major cause of blindness in third world countries. The ability of this obligate intracellular parasite to enter a non-phagocytic epithelial cell and survive within the hostile intracellular environment of the eukaryotic cytoplasm is key to its pathogenesis. The intimate interactions between chlamydia and its eukaryotic host is likely to involve natural biological pathways of the eukaryotic cell that the parasite usurps for its own survival. Study of these processes will yield insights into eukaryotic cell biology as well as insights into chlamydial disease pathogenesis. From these studies may emerge new therapeutic approaches to treating or preventing chlamydial infections.
Specific Aim 1 : The investigators hypothesize that successful C. trachomatis biovar LGV entry and intracellular development in epithelial cells involves at least two separate pathways, one of which is clathrin-independent, and have preliminary evidence that entry and/or development is dependent upon the host actin cytoskeleton and is modulated by c-src. (A) They will test the role of clathrin mediated endocytosis by assessing the effect in epithelial cells of expression of dominant negative (DN) alleles of dynamin, ARF-6, or clathrin on C trachomatis binding, entry, and replication. (B) They will further investigate the role of the actin cytoskeleton in the C trachomatis life cycle by determining whether the actin-regulating GTPases rac, rho, and CDC42 affect LGV and serovar E binding, entry, and replication in polarized and non-polarized epithelial cells. (C) They will determine the mechanism of c-src-mediated stimulation of C. trachomatis infectivity.
Specific aim 2 : An unusual aspect of the C. trachomatis life cycle is the receipt of sphingomyelin from the trans Golgi Network (TGN) by the bacteria-containing vacuole. They will test the hypothesis that the C. trachomatis vacuole interacts with one or more apical exocytic pathways including the newly proposed exocytic pathway in which lipid rafts transport sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and other designated proteins to the apical surface of polarized epithelial cells. Using several approaches, they will identify specific host cell factors required for the delivery of sphingomyelin from the TGN to the C. trachomatis vacuole. This will help to further define the pathway involved. These studies may lead to the development of new anti-chlamydial drug therapies and further our understanding of lipid trafficking in eukaryotic cells.
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