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

项目摘要 在美国，艰难梭菌感染每年侵袭近50万人， 导致近3万人死亡疾病预防控制中心宣布这种微生物对公共卫生构成“紧急”威胁， 最高威胁级别。迫切需要新的治疗方法。在这里，我们专注于C。难分辨细胞被膜 因为它是抗微生物治疗的常见靶点（如包括青霉素的抗生素所证明的）， 万古霉素和杆菌肽以及先天免疫系统的组分。梭难分辨细胞被膜 有一些不寻常的特征，可能被证明是抗生素的独特目标。溶菌酶通过以下方式杀死细胞 切割肽聚糖。我们分离了C.艰难梭菌对溶菌酶的抗性增加， 确定了两个调节系统，当组成型活性增加溶菌酶抗性。梭艰难 基因组包含相对大量的双组分系统，其中很少有研究。是 重要的是要了解C。difficile感知细胞外压力， 控制细胞膜的生物发生。我们将追求两个具体目标，以了解这些作用 调节因子在控制细胞包膜生物发生中的作用。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和功能突变的丧失。这些突变体将被研究， 它们对许多细胞被膜应激的抗性的作用和对细胞被膜生物发生的作用。这 将提供对这些双组分系统的表型效应的更好理解。在目标2中， 我们将绘制我们已经确定的一些基因的启动子，这些基因的表达依赖于这些启动子。 监管部门我们还将使用RNA-seq定义两种反应调节因子的调节子。最后，我们将使用 CRISPR干扰和过表达以鉴定溶菌酶抗性和细胞增殖所需的单个基因 包络线维护这些目标将促进我们对C的理解。通过定义生物学 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.