Genetic Analysis in Chlamydia

描述(申请人提供)：专性细胞内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上皮表面。反复和持续感染引起的慢性炎症可能会导致严重的并发症，如致盲沙眼、盆腔炎和不孕症。尽管衣原体疾病对临床和公共卫生具有重要意义，但这种细菌对突变分析的顽固性大大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基因分析的方法。在目标1中，我们将通过与参考菌株回交突变和单核苷酸多态(SNP)与SPQ表型的共分离来鉴定导致“小斑块”(SPQ)表型形成的突变。然后，通过对与SPQ表型共分离的DNA最小区域进行测序，确定可能的致病突变。在AIM2中，我们将定义衣原体在组织培养细胞中复制所需的必要基因集。来自一大套独立衍生的、高度诱变的衣原体分离株的染色体DNA将在池中进行测序。我们将通过监测单个基因耐受功能丧失突变的能力来评估单个基因对衣原体生存的相对贡献。建议的方法将大大加快衣原体基因组序列向功能信息的转换，并为其他“遗传难治”病原体的遗传分析建立蓝图。 与公共卫生的相关性：衣原体感染是一个重大的公共卫生负担，是发展生殖疾病的高风险。由于缺乏进行基因分析的工具，我们对它们的生物学和它们如何导致疾病的了解受到限制。在这里，我们建议开发基于新的DNA测序技术的方法来执行这样的分析。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.