Genetic Analysis in Chlamydia

描述（由申请人提供）：专性细胞内细菌沙眼衣原体是一种广泛传播的病原体，感染结膜和泌尿生殖道的上皮表面。反复和持续感染的慢性炎症可导致严重的并发症，如致盲性沙眼、盆腔炎和不孕症。尽管衣原体疾病的临床和公共卫生的重要性，细菌的突变分析的棘手性，显着阻碍了其发病机制的分子理解。在这项资助申请中，我们建议开发一种使用化学诱变剂和DNA深度测序技术进行衣原体遗传分析的方法。在目标1中，我们将通过将突变体与参考菌株回交并遵循单核苷酸多态性（SNP）与Spq表型的共分离来鉴定负责形成“小斑块”（Spq）表型的突变。然后通过对与Spq表型共分离的DNA最小区域进行测序来鉴定推定的致病突变。在目标2中，我们将定义衣原体在组织培养细胞中复制所需的基本基因集。将在样本池中对大量独立来源的重度诱变衣原体分离株的染色体DNA进行测序。我们将通过监测单个基因对功能丧失突变的耐受能力来评估其对衣原体生存能力的相对贡献。所提出的方法将显著加速衣原体基因组序列转化为功能信息，并为其他“遗传难治性”病原体的遗传分析建立蓝图。 公共卫生相关性：衣原体感染是一个重大的公共卫生负担，是生殖系统疾病发展的高风险。由于缺乏进行遗传分析的工具，我们对它们的生物学以及它们如何引起疾病的理解受到限制。在这里，我们建议开发方法来执行这样的分析的基础上新的DNA测序技术。这项研究的结果将确定衣原体在宿主中存活的重要因素，并为治疗干预和疫苗设计提供新的靶点。

DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated pathogen that infects epithelial surfaces of the conjuctiva and urogenital tract. Chronic inflammation from repeated and persistent infections can lead to severe complications such as blinding trachoma, pelvic inflammatory disease and infertility. Despite the clinical and public health importance of chlamydial diseases, the bacterium's intractability to mutational analysis has significantly hindered a molecular understanding of its pathogenesis. In this grant application, we propose to develop a methodology using chemical mutagens and DNA deep sequencing technologies to perform genetic analysis in Chlamydia. In Aim 1, we will identify mutations responsible for the formation of a "small plaque" (Spq) phenotype by backcrossing mutants to reference strains and following the co-segregation of single nucleotide polymorphisms (SNP) with the Spq phenotype. Putative causative mutations will be then identified by sequencing the minimal region of DNA that co-segregates with the Spq phenotype. In Aim2, we will define the essential gene set required for Chlamydia replication in tissue culture cells. Chromosomal DNA from a large set of independently derived, heavily mutagenized Chlamydia isolates will be sequenced in pools. We will assess the relative contribution of individual genes to Chlamydia viability by monitoring their ability to tolerate loss-of-function mutations. The proposed approaches will significantly accelerate the translation of Chlamydia genomic sequences into functional information and establish a blueprint for genetic analysis in other "genetically-intractable" pathogens. PUBLIC HEALTH RELEVANCE: Chlamydial infections are a significant public health burden and a high risk for the development of reproductive diseases. Our understanding of their biology and how they cause disease is limited by the lack of tools to perform genetic analysis. Here we propose to develop methods to perform such an analysis based on new DNA sequencing technologies. Results from this research will identify factors important for Chlamydia survival in the host and provide new targets for therapeutic intervention and vaccine design.