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Development and Validation of Conditional Gene Expression Systems in Chlamydia

衣原体条件基因表达系统的开发和验证

基本信息

批准号：
8605516
负责人：
P Scott Hefty
金额：
$ 18.29万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-15 至 2015-12-31
项目状态：
已结题

项目摘要

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.

描述(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。尽管沙眼衣原体对公众健康造成了巨大的影响，但人们对沙眼衣原体的基础生物学、遗传学和致病机制等许多方面都知之甚少。这反映了衣原体研究的一个主要局限性：缺乏系统进行定向基因操作。最近报道了两项重要的成果：一是将DNA导入衣原体的方法(转化)，二是开发出在大肠杆菌和衣原体中都稳定的带有有效选择的抗生素标记的质粒(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将所需的DNA序列直接引入衣原体，评估由此产生的生物效应，定义毒力成分，并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有针对性的基因抑制对于适当分析衣原体特定蛋白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。许多实现基因表达和抑制的分子成分已经成功地应用于模型细菌系统；然而，还没有在衣原体中进行测试。提出了三个具体的目标：1)条件基因表达，2)靶向基因抑制，3)荧光蛋白验证。开发这些针对衣原体的分子工具将是衣原体领域的关键成就。此外，这将首次允许直接证明和定义衣原体致病机制的能力。