Identification of a novel two-component system involved in peptidoglycan synthesis in Clostridioides difficile

摘要 艰难梭状芽胞杆菌是一种芽胞形成的厌氧细菌，可导致严重的疾病，包括 人类抗生素相关性腹泻和伪膜性结肠炎。万古霉素是治疗癌症的一线药物 治疗艰难梭菌感染；它的靶点是肽聚糖的生物合成，这是原核细胞和 对细菌细胞壁的形成和生长是必不可少的。万古霉素与D-丙氨酸-D-丙氨酸残基的结合 并阻止它们与成熟的肽多聚糖结合。 艰难梭菌的Vang操纵子，其类似物使其他细菌对万古霉素产生抗药性 由于肽聚糖的D-丙氨酸-D-丙氨酸部分被D-丙氨酸-D-丝氨酸取代，受 一个双组分系统，VanRS。独特的是，既不是万古霉素诱导的，也不是高水平的，结构性表达 艰难梭菌中的Vang操纵子本身对万古霉素具有耐药性。尽管如此，许多临床和 实验室产生的万古霉素耐药艰难梭菌含有VanRS突变，使Vang增加 表达，强烈表明操纵子的高表达与其他突变一起， 以促进抵抗运动的发展。 我们已经发现，在缺乏艰难梭菌对万古霉素敏感的组氨酸激酶的情况下， 另一种尚未从基因上鉴定并暂时命名为KinX的组氨酸激酶也对 万古霉素，并能够取代面包车和诱导Vang操纵子。一种受调控的组氨酸激酶串扰 对同样的环境信号的反应，在这种情况下，万古霉素，是不寻常的。与面包车不同，KinX还 对至少另一种干扰肽聚糖合成的抗生素有反应。因此，它是至关重要的 详细了解KinX的功能很重要，它是在临床使用时激活的 抗生素，可能通过调节Vang而导致艰难梭菌对万古霉素的耐药性。 操纵子，并且很可能调节与肽聚糖新陈代谢有关的额外基因。 使用几种独立、公正或有针对性的方法，包括RNA-Seq和CRISPRi，我们 建议确定新的组氨酸激酶，KinX，以及可能控制其同源反应调节因子 肽聚糖生物合成相关基因的表达。使用特定基因和全球表达分析，我们 将确定KinX对调节Vang操纵子的贡献，并定义KinX调节子。我们的 这些结果将为研究肽聚糖的生物合成和万古霉素敏感性及致病机制提供新的线索 艰难梭菌的耐药性。 对万古霉素耐药的菌株在临床上很常见，耐药性的传播可能 成为治疗艰难梭菌感染的一个严重问题。关于VANG规则的详细知识 操纵子和其他肽聚糖代谢基因对于理解多肽多糖的发育至关重要。 万古霉素耐药性和设计针对肽聚糖的新抗菌剂。

ABSTRACT Clostridioides difficile is a spore-forming, anaerobic bacterium that can cause severe disease, including antibiotic-associated diarrhea and pseudomembranous colitis, in humans. Vancomycin is a first-line drug for treating C. difficile infection; it targets peptidoglycan biosynthesis, a pathway specific for prokaryotic cells and essential for the formation of the bacterial cell wall and growth. Vancomycin binds to the D-Ala-D-Ala residues of the peptidoglycan intermediates and prevents their incorporation into mature peptidoglycan. The C. difficile vanG operon, analogs of which confer vancomycin resistance in other bacterial species due to the replacement of the D-Ala-D-Ala moiety of peptidoglycan with D-Ala-D-Ser, is positively regulated by a two-component system, VanRS. Uniquely, neither vancomycin-induced nor high, constitutive expression of the vanG operon confers by itself resistance to vancomycin in C. difficile. Nevertheless, many clinical and laboratory-generated vancomycin-resistant C. difficile strains contain vanRS mutations that increase vanG expression, strongly suggesting that high expression of the operon contributes, together with other mutations, to the development of the resistance. We have found that in the absence of the C. difficile vancomycin-sensing histidine kinase, VanS, another histidine kinase, not yet genetically identified and provisionally named as KinX, also responds to vancomycin and is able to replace VanS and induce the vanG operon. A regulated histidine kinase crosstalk in response to the same environmental signal, in this case vancomycin, is unusual. In contrast to VanS, KinX also responds to at least one more antibiotic that interferes with peptidoglycan synthesis. Therefore, it is critically important to understand in detail the function of KinX, which is activated in response to a clinically used antibiotic, may contribute to the emerging resistance of C. difficile to vancomycin via the regulation of the vanG operon, and is very likely to regulate additional genes that are involved in peptidoglycan metabolism. Using several independent unbiased or targeted approaches, including RNA-Seq and CRISPRi, we propose to identify the novel histidine kinase, KinX, and, likely, its cognate response regulator that control expression of genes of peptidoglycan biosynthesis. Using gene-specific and global expression analyses, we will determine the contribution of KinX to the regulation of the vanG operon and define the KinX regulon. Our results will shed new light on peptidoglycan biosynthesis and mechanisms of vancomycin sensitivity and resistance in C. difficile. Vancomycin-resistant strains are commonly detected in the clinic, and the spread of the resistance may become a serious issue in treating C. difficile infection. Detailed knowledge on the regulation of the vanG operon and other genes of peptidoglycan metabolism is critical for understanding the development of vancomycin resistance and designing new antimicrobials that target peptidoglycan.