Analysis of chlamydial recombination in vivo

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DESCRIPTION (provided by applicant): Recent work in our and other laboratories has demonstrated the chlamydiae to be actively recombinogenic. This supports previous nucleotide sequence analysis supporting intraspecies recombination in this system. We have a long-term goal of understanding the mechanisms of genetic exchange by chlamydiae, particularly as it may occur in vivo. We also are pursuing the use of recombinant strains as tools to examine chlamydial gene function, as these organisms are not amenable to targeted gene introduction or disruption. To address these goals, we propose to explore chlamydial recombination in a mouse model of chlamydial genital infection. Our research group has identified or selected for antibiotic resistant strains of Chlamydia muridarum, C. trachomatis, and C. suis, all of which are recombinogenic in vitro. We will use these strains in experiments to accomplish the following Aims. First, we will test the hypothesis that chlamydiae can recombine in vivo by inoculating pairs of differently antibiotic resistant chlamydial strains into mice that will then be treated with antibiotics that will select for recombinants. Conditions will be optimized to recover chlamydiae that grow in these antibiotic treated mice, and their genomes will be sequenced to determine the nature of recombination in this system. The second aim of the proposal will use in vitro generated recombinants to examine gene function in host tropism by different, but highly related, chlamydial species. The murine pathogen C. muridarum will be crossed in vitro with a strain of the human pathogen C. trachomatis, and a set of independent recombinants will be cloned and characterized in vitro. The genomes will be sequences for a set of these progeny, and a subset (approx. 12) of these variable strains will be inoculated into mice. Chlamydial development will be assessed by culture and histological analysis of these infected animals. Bioinformatics analyses will then be used to associate chlamydial genotype with the phenotypes identified in vitro and in vivo. We anticipate that these studies will identify genes or gene sets that are associated with phenotypic variability between the species. PUBLIC HEALTH RELEVANCE: Infections by different chlamydial species leads to serious human diseases worldwide, including blinding trachoma, pelvic inflammatory disease, and infertility. While there is no practical genetic system yet available to study gene function and virulence properties, chlamydiae recombine actively in vitro. This is an interesting phenomenon that may be a useful tool for studying chlamydial gene function. In this proposal we will use an in vivo model to explore both the mechanisms and significance of recombination in vivo, and to explore gene function in an animal model system. These studies should help identify candidate proteins that can be targeted for novel immunogenic or therapeutic purposes, and address the question of the significance of recombination in the chlamydial system in vivo.