Regulation of the C. difficile cell envelope by Two-component systems

项目摘要 在美国，艰难梭菌感染每年造成近50万人死亡， 导致近3万人死亡。美国疾病控制与预防中心已宣布这种微生物对公共健康构成“紧急”威胁。 最高威胁等级。迫切需要新的治疗方法。在这里，我们主要关注艰难梭菌的细胞膜 因为它是包括青霉素在内的抗生素所证明的抗菌治疗的常见靶点， 万古霉素和杆菌素以及先天免疫系统的组成部分。艰难梭菌的细胞膜 有一些不寻常的特征，可能会被证明是抗生素的独特靶点。杀灭细胞的溶菌酶 裂解肽多糖。我们分离了艰难梭菌对溶菌酶的抗性增加的突变株，我们 确定了两个调节系统，当结构性活跃时，会增加溶菌酶的抗性。艰难梭菌 基因组包含数量相对较多的两组分系统，但很少有人对其进行研究。它是 重要的是要了解艰难梭菌如何感知细胞外压力以及这些调节系统如何 控制细胞包膜的生物发生。我们将追求两个具体目标，以了解这些 调控细胞被膜生物发生的调节剂。在目标1中，我们将构建一系列具有 这些调节系统中的功能突变和功能缺失。然后将对这些突变体进行研究 它们对一些细胞膜压力的抗性和对细胞膜生物发生的影响。这 将有助于更好地理解这些双组分系统的表型效应。在《目标2》中，我们 将映射一些我们已经确定的基因的启动子，这些基因的表达依赖于这些 监管者。我们还将使用RNA-seq来定义这两个反应调节器的调节子。最后，我们将使用 CRISPR干扰和过表达以识别溶菌酶抗性和细胞所需的单个基因 信封维护。这些目标结合在一起将通过定义困难梭菌的 两种不同的双组分系统在控制细胞包膜维持中的作用。

Project Summary Clostridioides (Clostridium) difficile infections strike close to 500,000 people a year in the United States, leading to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest threat category. New treatments are sorely needed. Here we focus on the C. difficile cell envelope because it is a common target of antimicrobial therapy as evidenced by antibiotics including penicillin, vancomycin and bacitracin as well as components of the innate immune system. The C. difficile cell envelope has some unusual features that may prove to be unique targets for antibiotics. Lysozyme which kills cells by cleaving the peptidoglycan. We isolated mutants of C. difficile with increased resistance to lysozyme and we identified two regulatory systems that when constitutively active increase lysozyme resistance. The C. difficile genome contains a relatively large number of two component systems of which few have been studied. It is important to understand how C. difficile senses extracellular stresses and how these regulatory systems control biogenesis of the cell envelope. We will pursue two specific aims to understand the role of these regulators in controlling cell envelope biogenesis. In Aim 1 we will construct a series of mutants that have gain of function and loss of function mutations in these regulatory systems. These mutants will then be studied for their effect on resistance to a number of cell envelope stresses and effects on cell envelope biogenesis. This will provide a greater understanding of the phenotypic effects of these two-component systems. In Aim 2 we will map the promoters of some genes we have identified whose expression is dependent upon these regulators. We will also define the regulons of both response regulators using RNA-seq. Finally, we will use CRISPR interference and overexpression to identify individual genes required for lysozyme resistance and cell envelope maintenance. Together these aims will advance our understanding of C. difficile biology by defining the role of two different two-component system in controlling cell envelope maintenance.