Forward and Reverse Genetics in Chlamydia

描述（由申请人提供）：使用分子遗传工具在致病菌中产生和补充突变是我们理解微生物如何引起疾病的核心。不幸的是，许多临床上重要的病原体，如沙眼衣原体-致盲沙眼和许多性传播疾病的病原体-不适合这种分析。在这里，我们建议建立一个全面的系统，在衣原体的正向和反向遗传分析的基础上使用化学诱变剂，遗传重组和“深度”DNA测序技术。我们的具体目标是：1）确定C中的重要因素。通过实施正向遗传筛查，包括II型分泌物的遗传分析，研究沙眼衣原体的发病机制; 2）开发用于沙眼衣原体系统性反向遗传分析的方法。沙眼 通过完成这些实验，我们将建立方法来进行常规的正向和遗传筛选-微生物致病机理研究的关键。通过拟议的工作产生的工具和试剂将构成衣原体研究界的重要资源。 公共卫生相关性：沙眼衣原体是传染性失明（沙眼）和许多性传播感染的主要原因。尽管其临床和公共卫生的重要性，这些专性细胞内细菌病原体仍然研究不足，因为它们的棘手的分子遗传操作。在这里，我们描述了在这些病原体中进行遗传分析的方法，这将大大加快我们对毒力基因功能的理解和减毒菌株用于疫苗应用的潜在发展。

DESCRIPTION (provided by applicant): The use of molecular genetic tools to generate and complement mutations in pathogenic bacteria is central to our understanding of how microorganisms cause disease. Unfortunately, many clinically important pathogens, such as Chlamydia trachomatis -the causative agent of blinding trachoma and many sexually transmitted diseases- are not amenable to such analysis. Here we propose to establish a comprehensive system for forward and reverse genetic analysis in Chlamydia based on the use of chemical mutagens, genetic recombination and "deep" DNA sequencing technologies. Our specific goals are to 1) identify factors important in C. trachomatis pathogenesis by implementing forward genetic screens, including a genetic analysis of Type II secretion and 2) develop a method for systematic reverse genetic analysis in C. trachomatis. By the completion of these experiments we will have established methods to perform routine forward and genetic screens - the lynchpin of microbial pathogenesis research. The tools and reagents generated through the proposed work will constitute an important resource for the Chlamydia research community. PUBLIC HEALTH RELEVANCE: Chlamydia trachomatis is the leading cause of infectious blindness (trachoma) and many sexually transmitted infections. Despite their clinical and public health importance, these obligate intracellular bacterial pathogens remain understudied because of their intractability to molecular genetic manipulation. Here we describe methods to perform genetic analysis in these pathogens, which will significantly accelerate our understanding of virulence gene function and the potential development of attenuated strains for vaccine applications.