Analysis of chlamydial recombination in vivo

描述(由申请人提供)：我们和其他实验室最近的研究表明，衣原体是主动重组的。这支持了以前的核苷酸序列分析，支持该系统中的种内重组。我们有一个长期的目标，即了解衣原体基因交换的机制，特别是在体内可能发生的情况下。我们还在寻求使用重组菌株作为检查衣原体基因功能的工具，因为这些生物不受靶向基因导入或破坏的影响。为了解决这些目标，我们建议在衣原体生殖道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筛选出耐药的沙眼衣原体、沙眼衣原体和猪链球菌，它们都是体外重组的。我们将在实验中使用这些菌株来实现以下目标。首先，我们将通过将一对不同抗药性的衣原体菌株接种到小鼠体内来检验衣原体可以在体内重组的假设，然后用抗生素治疗这些菌株，以选择重组子。将对条件进行优化，以恢复在这些抗生素处理的小鼠中生长的衣原体，并对它们的基因组进行测序，以确定该系统中重组的性质。该提案的第二个目的将使用体外产生的重组体来检查不同但高度相关的衣原体物种在宿主趋向性方面的基因功能。小鼠病原体将在体外与人类病原体沙眼衣原体杂交，并将克隆一组独立的重组体，并在体外对其进行鉴定。基因组将是这些子代的一组序列，以及一个子集(约12)这些变异菌株中的一种将被接种到老鼠体内。衣原体的发育将通过对这些受感染动物的培养和组织学分析进行评估。然后将使用生物信息学分析将衣原体基因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的表型变异相关的基因或基因集。 公共卫生相关性：不同衣原体感染可导致世界范围内严重的人类疾病，包括致盲沙眼、盆腔炎和不孕症。虽然目前还没有实用的遗传系统来研究基因功能和毒力特性，但衣原体在体外进行了积极的重组。这是一个有趣的现象，可能是研究衣原体基因功能的有用工具。在这个方案中，我们将使用体内模型来探索体内重组的机制和意义，并在动物模型系统中探索基因功能。这些研究应该有助于确定可用于新的免疫原性或治疗目的的候选蛋白质，并解决体内衣原体系统中重组的意义问题。

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.