Development of a novel protein depletion method in Chlamydia

项目摘要/摘要 超过180万例沙眼衣原体感染病例被报告给 美国疾病控制与预防中心每年都会出现这种疾病，使其成为该国报告最常见的传染病。 这种病原体是一种专有的细胞内细菌，只能在人类体内繁殖。 细胞。由于对宿主细胞的这种依赖，衣原体经历了还原 进化，拥有最小的细菌基因组之一。剩下的许多人 基因，包括衣原体特异性基因，是必需的基因，其功能有 由于目前遗传方法的局限性，这一点尚未阐明。学习 衣原体基因功能，我们正在开发一种新的蛋白质耗尽方法， 利用小RNA(SRNAs)下调特定靶标表达的能力 蛋白质。在原理证明实验中，我们已经证明了我们可以归纳地 两种衣原体蛋白Inca和IncG的击倒蛋白水平。在目标1中，我们将 敲除其他沙眼衣原体基因，包括非必需基因和必需基因 基因。在目标2中，我们将把这种sRNA敲除方法与另一种蛋白质进行比较。 耗竭法，CRISPRi。我们将比较每种方法控制 击倒的水平和时间，并将调查它们是否对 同一操纵子中的基因。成功完成这些研究将导致 选择性消耗衣原体蛋白的创新遗传方法。这个新工具 对衣原体基因功能的研究将对该领域产生持续的影响。

Project Summary/Abstract More than 1.8 million cases of Chlamydia trachomatis infections are reported to the CDC each year, making it the most commonly reported infectious disease in the country. This pathogen is an obligate intracellular bacterium that can only reproduce in human cells. As a result of this dependence on a host cell, Chlamydia has undergone reductive evolution and has one of the smallest of bacterial genomes. Many of the remaining genes, including Chlamydia-specific genes, are essential genes whose functions have not been elucidated because of the limitations of current genetic methods. To study chlamydial gene function, we are developing a novel protein depletion method that exploits the ability of small RNAs (sRNAs) to downregulate expression of specific target proteins. In proof-of-principle experiments, we have shown that we can inducibly knockdown protein levels for two chlamydial proteins, IncA and IncG. In Aim 1, we will knockdown additional C. trachomatis genes, including non-essential and essential genes. In Aim 2, we will compare this sRNA knockdown method to another protein depletion method, CRISPRi. We will compare the ability of each method to control the level and timing of knockdown and will investigate whether they cause polar effects on genes within the same operon. Successful completion of these studies will lead to an innovative genetic approach for selectively depleting a Chlamydia protein. This new tool for studying chlamydial gene function will have a sustained impact on the field.