Development and Validation of Conditional Gene Expression Systems in Chlamydia

描述（由申请人提供）：沙眼衣原体是困扰人类的最重要的细菌病原体之一。尽管这对公众健康有巨大的影响，但关于C。对沙眼的基本生物学、遗传学和发病机制了解甚少。这反映了衣原体研究的主要局限性：缺乏一个系统 进行定向基因操作最近报道了两项重要的成就：将DNA导入衣原体的方法（转化）和在两种大肠杆菌中都稳定的质粒的开发。大肠杆菌和衣原体的有效选择性抗生素标记（穿梭载体）。这种方法和工具可以使我们在衣原体领域第一次能够 直接将所需的DNA序列导入衣原体，并评估产生的生物学效应，定义毒力成分，更好地了解疾病是如何引起和改善的。严格控制基因表达和靶向基因抑制对于适当分析衣原体中给定蛋白质的生物学效应至关重要，特别是考虑到感染和发育的多个阶段。许多分子组件，以实现受控的基因表达和镇压已成功地用于模式细菌系统，但没有在衣原体测试。提出了三个具体目标：1）条件基因表达，2）靶向基因抑制，3）荧光蛋白验证。在衣原体中开发这些分子工具将是衣原体领域的关键成就。此外，将允许第一次直接证明和定义衣原体发病机制的能力。

DESCRIPTION (provided by applicant): Chlamydia trachomatis is among the most significant bacterial pathogens afflicting humans. Despite this enormous impact on public health, many aspects about C. trachomatis including basic biology, genetics, and pathogenesis are poorly understood. This is reflective of a primary limitation in chlamydial research: the lack of a system for directed genetic manipulation. Two significant achievements were recently reported: a method for introducing DNA into Chlamydia (transformation) and development of a plasmid stable in both E. coli and Chlamydia with an effective selectable antibiotic marker (shuttle vector). This method and tool can enable us, for the first time in the Chlamydia field, the ability to directly introduce desired DNA sequences into Chlamydia and evaluate resulting biological effects, define virulence components, and better understand how disease is caused and ameliorated. Tightly controlled gene expression and targeted gene repression is essential to appropriately analyze the biological effect of a given protein in Chlamydia, especially given the many stages of infection and development. Many molecular components to achieve controlled gene expression and repression have been employed successfully in model bacterial systems; however, none have been tested in Chlamydia. Three specific aims are proposed; 1) Conditional gene expression, 2) Targeted gene repression, and 3) Fluorescent protein validation. Developing these molecular tools in Chlamydia would be pivotal accomplishments in the chlamydial field. Moreover, would allow for the first time the ability to directly demonstrate and define mechanisms for chlamydial pathogenesis.