Analysis of chlamydial recombination in vivo

描述（由申请人提供）：最近在我们和其他实验室的工作已经证明衣原体是积极重组的。这支持了先前的核苷酸序列分析，支持该系统中的种内重组。我们的长期目标是了解衣原体的遗传交换机制，特别是它可能发生在体内。我们也在寻求使用重组菌株作为工具来检查衣原体基因功能，因为这些生物体不适合靶向基因导入或破坏。为了实现这些目标，我们建议在衣原体生殖器感染的小鼠模型中探索衣原体重组。我们的研究小组已经鉴定或选择了耐药菌株muridarum衣原体，沙眼衣原体和猪衣原体，所有这些菌株都是体外重组的。我们将在实验中使用这些菌株来达到以下目的。首先，我们将验证衣原体可以在体内重组的假设，方法是将对不同抗生素耐药的衣原体菌株接种到小鼠体内，然后用抗生素治疗小鼠，抗生素将选择重组菌株。将优化条件以恢复在这些抗生素治疗小鼠中生长的衣原体，并对其基因组进行测序以确定该系统中重组的性质。该提案的第二个目的是利用体外生成的重组体来检测不同但高度相关的衣原体物种在宿主趋向性中的基因功能。将鼠源沙眼衣原体与人源沙眼衣原体进行体外杂交，克隆出一组独立的重组体并进行体外鉴定。基因组将是这些后代中的一组的序列，以及一个子集(大约。将这些变异菌株中的12种接种到小鼠体内。将通过对这些感染动物的培养和组织学分析来评估衣原体的发育情况。然后，生物信息学分析将用于将衣原体基因型与体外和体内鉴定的表型联系起来。我们预计这些研究将确定与物种之间表型变异相关的基因或基因集。

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.