Development of a novel protein depletion method in Chlamydia

项目总结/摘要 向世界卫生组织报告的沙眼衣原体感染病例超过180万例， 疾病控制和预防中心每年，使其成为最常见的报告传染病在该国。 这种病原体是一种专性细胞内细菌，只能在人体内繁殖 细胞由于这种对宿主细胞的依赖性，衣原体经历了还原性生长， 它是最小的细菌基因组之一其余许多 基因，包括衣原体特异性基因，是必需的基因，其功能具有 由于目前遗传学方法的局限性，尚未阐明。研究 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）下调特定靶点表达的能力， proteins.在原理验证实验中，我们已经表明，我们可以诱导 两种衣原体蛋白印加和IncG的敲低蛋白水平。在目标1中，我们 击倒额外的C。沙眼基因，包括非必需基因和必需基因 基因.在目标2中，我们将比较这种sRNA敲除方法与另一种蛋白质 耗尽方法，CRISPRi.我们将比较每种方法控制 水平和时间的击倒，并将调查他们是否会造成极地影响， 同一操纵子中的基因。成功完成这些研究将导致 创新的遗传方法选择性地消耗衣原体蛋白质。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.