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，可感染结膜和泌尿生殖道上皮表面。反复和持续感染引起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。尽管衣原体疾病在临床和公共卫生方面具有重要意义，但这种细菌难以进行突变分析，这极大地阻碍了对其发病机制的分子理解。在这项拨款申请中，我们建议开发一种使用化学诱变剂和DNA深度测序技术来进行衣原体遗传分析的方法。在Aim 1中，我们将通过将突变体与参考菌株回交并将单核苷酸多态性（SNP）与Spq表型共分离，确定负责形成“小斑块”（Spq）表型的突变。假定的致病突变将通过测序与Spq表型共分离的DNA的最小区域来确定。在ai2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。从大量独立衍生的、严重诱变的衣原体分离物中提取的染色体DNA将在池中测序。我们将通过监测个体基因耐受功能丧失突变的能力来评估个体基因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“遗传难以处理”的病原体的遗传分析建立蓝图。

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.