Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the cause of a wide spectrum of human diseases. Immunopathology caused from repeated and persistent infection mediates the most severe disease outcomes. Chlamydia have a complex biphasic developmental cycle. Two developmental forms have been characterized; the infectious and metabolically inactive elementary body (EB) and the noninfectious vegetative form, the reticulate body (RB). The differentiation to one form or the other involves the coordinate control of gene expression. This hypothesis is supported by inhibition studies, Northern hybridization studies and identification of developmental-stage specific gene products. The long-term objective is to understand the biochemical and molecular mechanisms of developmental regulation of these organisms. This will yield important fundamental information for a) developing cell-free growth of these organisms, b) characterizing developmentally regulated components that mediate pathogenesis and virulence, c) understanding the mechanisms of persistent infection, d) expression of chlamydial genes directly in E. coli, and e) identification of unique targets for the design of new chemotherapeutic agents.
The specific aims will define microbiologic mechanisms involved with the temporal and sequential regulation of C. trachomatis development at the level of transcription of specific genes. These are: 1) Clone developmentally regulated genes and evaluate and compare regulatory 5' flanking sequences to delineate those sequences responsible for gene selectivity by chlamydial RNA polymerase. Gene products will also be available for study. Some genes will be identified by differential hybridization of cDNA representing different stages of development. 2) Characterize chlamydial RNA polymerase sigma subunits and clone their respective genes. Although gene regulation is complex, the focus will be on the identification of sigma subunits because these determine the specificity for developmental-specific promoter families. Polymerase subunits will be identified by immunoprecipitation of holoenzyme. Polymerase subunit genes will be identified in recombinant libraries by either gene probes or by immunodetection. 3) Functional studies using the materials developed from aims 1&2 to characterize polymerase cognate promoter sequences. Reconstitution of subunits and binding to promoter sequences will link specific sigma subunits with chlamydial consensus promoter sequences. These may then be used to detect additional developmental-specific genes by binding selection in vitro or by shotgun cloning in E. coli carrying chlamydial polymerase genes.
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