Development of a novel protein depletion method in Chlamydia

项目概要/摘要 已报告沙眼衣原体感染病例超过 180 万例 疾病预防控制中心每年都会报告这种疾病，使其成为该国最常报告的传染病。 该病原体是一种专性细胞内细菌，只能在人体中繁殖 细胞。由于这种对宿主细胞的依赖性，衣原体经历了还原 进化并具有最小的细菌基因组之一。剩下的很多 基因，包括衣原体特异性基因，是必需基因，其功能 由于目前遗传学方法的局限性，尚未得到阐明。学习 衣原体基因功能，我们正在开发一种新的蛋白质消耗方法 利用小 RNA (sRNA) 下调特定靶标表达的能力 蛋白质。在原理验证实验中，我们已经证明我们可以归纳 两种衣原体蛋白 IncA 和 IncG 的敲低蛋白水平。在目标 1 中，我们将 敲低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，我们将这种 sRNA 敲低方法与另一种蛋白质进行比较 耗尽方法，CRISPRi。我们将比较每种方法控制 击倒的水平和时间，并将调查它们是否会造成极性影响 同一操纵子内的基因。成功完成这些研究将导致 用于选择性消耗衣原体蛋白的创新遗传方法。这个新工具 对于研究衣原体基因功能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.