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

项目概要 在美国，每年有近 50 万人感染艰难梭菌， 导致近3万人死亡。疾病预防控制中心宣布这种生物体对公众健康构成“紧急”威胁 最高威胁类别。迫切需要新的治疗方法。这里我们重点关注艰难梭菌细胞包膜 因为它是抗菌治疗的常见目标，包括青霉素在内的抗生素就证明了这一点， 万古霉素和杆菌肽以及先天免疫系统的成分。艰难梭菌细胞膜 具有一些不寻常的特征，可能被证明是抗生素的独特靶点。溶菌酶通过以下方式杀死细胞 裂解肽聚糖。我们分离了对溶菌酶具有增强抗性的艰难梭菌突变体，并且我们 确定了两个调节系统，当组成型活性时，它们会增加溶菌酶抗性。艰难梭菌 基因组包含相对大量的两个组成系统，但其中很少被研究。它是 了解艰难梭菌如何感知细胞外应激以及这些调节系统如何 控制细胞膜的生物发生。我们将追求两个具体目标来理解这些的作用 控制细胞被膜生物发生的调节因子。在目标 1 中，我们将构建一系列具有增益的突变体 这些调节系统的功能突变和功能丧失突变。然后将研究这些突变体 它们对抵抗多种细胞被膜应激的影响以及对细胞被膜生物发生的影响。这个 将提供对这些双组分系统的表型效应的更深入的了解。在目标 2 中，我们 将绘制我们已识别的一些基因的启动子图谱，这些基因的表达取决于这些基因 监管机构。我们还将使用 RNA-seq 定义两个反应调节器的调节子。最后，我们将使用 CRISPR 干扰和过度表达，以识别溶菌酶抗性和细胞所需的单个基因 信封维护。这些目标共同将通过定义艰难梭菌生物学来增进我们对艰难梭菌生物学的理解 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.