The long-term goal of this research is to contribute to the knowledge of the regulation of the chlamydial development cycle, as it relates specifically to latent or persistence of infection, by: (i) performance of structure/function studies of chlamydial elongation factors Tu (EF-Tu) and Ts (EF-Ts) to define their specific function in protein synthesis in a model system (ii) definition of the roles of these proteins and of their related factors in the growth, division and differentiation of Chlamydia trachomatis in cell culture in vitro; and (iii) performance of clinical studies using the knowledge obtained from in vitro studies for understanding of the mechanisms of growth and differentiation control in vivo.
Three specific aims are involved in the proposed studies.
SPECIFIC AIM 1 is to determine the three dimensional structures of chlamydial EF-Tu and EF-Ts molecules by X-ray crystallography, with mapping of their complete binding surfaces. Recombinant EF-Tu and EF-Ts will be over- expressed in E. coli and immuno-purified using monoclonal antibodies (MAbs). Crystallization and structure determination will be performed in Dr. Gregory A. Petsko's laboratory of Brandeis University. The structural data will be used to predict the precise location in the elongation factor molecules where we hypothesize modifications would have the greatest impact upon the functional characteristics of these molecules.
SPECIFIC AIM 2 is to assess the role of altered elongation factors utilizing complementation analysis, both in vitro and in vivo. An E. coli cell free coupled translation-transcription system will be employed for in vitro replacement analysis; extracts will be freed of E. coli elongation factors by immunoabsorption with MAbs specific for E. coli elongation factors. The level of DNA-directed translation will be used as a marker to assess the activities of replaced EF-Tu or EF-Ts. For in vivo replacement, the tufA/EC::Kan-tuf/CTF fragment (EC = E. coli, CTF = C. trachomatis serovar F) will be inserted into pHSG415, a low copy number plasmic containing a replication temperature sensitive control. The reconstructed plasmid will be transformed into E. coli HW123, an aurodox resistant strain that contains only a single copy of the EF-Tu gene to be replaced. The model system, once developed, will be used to study the regulation of protein synthesis and the control of chlamydial development.
SPECIFIC AIM 3 is to establish the importance of elongation factors in chlamydial growth, division, differentiation or latency, using mRNA analysis as a surrogate marker. The mRNA analysis will be performed in laboratory cell cultures, and, more importantly, in clinical specimens obtained from patients whose chlamydial infections are either active (acute disease) or quiescent (e.g., infertile women with no evidence of current active disease). Reverse transcription-polymerase chain reaction (RT-PCR) will be employed to quantitate the mRNAs of EF-Tu and EF-Ts of C. trachomatis from infected HeLa cells in different stages of chlamydial development cycle and in endometrial specimens taken from Chlamydia infected women. These data will provide useful insights into the mechanisms of clinical activity or latency of Chlamydia infection.
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