Analysis of chlamydial recombination in vivo

体内衣原体重组分析

• 批准号: 7885155
• 负责人: DANIEL D ROCKEY
• 金额: $ 21.93万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885155
• 关键词: Address; Animal Model; Animals; Antibiotic Resistance; Antibiotics; Base Sequence; Bioinformatics; Biological Models; Chlamydia; Chlamydia Infections; Chlamydia muridarum; Chromosomes; Communities; Data; Development; Environment; Family; Gene Targeting; Generations; Genes; Genetic; Genetic Recombination; Genital system; Genome; Genomics; Genotype; Goals; Growth; Human; Immune; In Vitro; Infection; Infertility; Interferon Type II; Laboratories; Mus; Nature; Nutrient; Organism; Parents; Patients; Pelvic Inflammatory Disease; Phenotype; Probability; Procedures; Process; Property; Proteins; Recombinants; Research; Resources; Sequence Analysis; Specificity; Swab; System; Testing; Therapeutic; Time; Trachoma; Tropism; Virulence; Work; base; gene function; genital infection; genome sequencing; human disease; immunogenic; in vitro testing; in vivo; in vivo Model; interest; mouse model; mutant; novel; pathogen; public health relevance; research study; resistant strain; tool

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.

描述（由申请人提供）：我们和其他实验室的最新工作已经证明衣原体是活跃的重组原。这支持先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是了解衣原体遗传交换的机制，特别是因为它可能发生在体内。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。为了解决这些问题，我们建议探讨衣原体生殖器感染的小鼠模型中的衣原体重组。本课题组已鉴定或筛选出耐药的鼠衣原体、C. trachomatis和C. suis，所有这些都是体外重组基因。我们将在实验中使用这些菌株来实现以下目标。首先，我们将测试衣原体可以在体内重组的假设，通过将对不同抗生素耐药的衣原体菌株植入小鼠体内，然后用抗生素治疗，以选择重组体。将优化条件以回收在这些抗生素处理的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嗜性中的基因功能。鼠病原体C.将在体外与人类病原体C.沙眼衣原体，并且一组独立的重组体将被克隆并在体外表征。基因组将是这些后代的一组序列，以及一个子集（大约）。12)将这些可变菌株接种到小鼠中。将通过这些感染动物的培养和组织学分析评估衣原体发育。然后将使用生物信息学分析将衣原体基因型与体外和体内鉴定的表型相关联。我们预计，这些研究将确定基因或基因集与物种之间的表型变异。 公共卫生关系：不同种类的衣原体感染在世界范围内导致严重的人类疾病，包括致盲性沙眼、盆腔炎和不孕症。虽然还没有实用的遗传系统来研究基因功能和毒力特性，但衣原体在体外重组活跃。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。在本研究中，我们将使用一个体内模型来探讨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。这些研究应有助于确定候选蛋白质，可以针对新的免疫原性或治疗目的，并解决的问题，在衣原体系统在体内重组的意义。