Chlamydia trachomatis is an obligate intracellular pathogen that is the leading cause of sexually transmitted diseases in the United States and the major cause of preventable blindness in third world countries. The ability of this parasite to enter non-professional phagocytic epithelial cells and survive within the intracellular environment of the eukaryotic cytoplasm is the key to its pathogenesis. It is hypothesized that successful interaction between chlamydia and its host is dependent on early de novo chlamydial protein synthesis and that these proteins play a role in the structural and functional remodeling of the inclusion membrane thereby enabling the evasion of host degradation pathways. However, the identities of these proteins and the molecular mechanisms of interaction are either unknown or poorly understood. This proposal is designed to identify chlamydial proteins involved in the remodeling of the inclusion membrane and to characterize their interactions with host cell proteins.
Specific Aim 1 proposes to identify these chlamydial proteins by metabolic labeling with stable isotopes followed by proteomic analysis using Multi-dimensional Protein Identification Technology (MudPIT).
Specific Aim 2 utilizes subcellular fractionation coupled with subtractive proteomics to select for chlamydial proteins localized to the cytoplasmic surface of the inclusion membrane. Finally, Specific Aim 3 will focus on the selected chlamydial proteins from Specific Aim 2 and generate recombinant fusion proteins to identify interacting host proteins in vitro.
