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

摘要 艰难梭菌是一种形成孢子的厌氧细菌，可引起严重疾病，包括 人类抗生素相关性腹泻和伪膜性结肠炎。万古霉素是一线药物 治疗艰难梭菌感染；它的目标是肽聚糖生物合成，这是原核细胞特有的途径 对于细菌细胞壁的形成和生长至关重要。万古霉素与 D-Ala-D-Ala 残基结合 肽聚糖中间体并防止它们掺入成熟的肽聚糖中。 艰难梭菌 vanG 操纵子，其类似物赋予其他细菌物种万古霉素抗性 由于肽聚糖的 D-Ala-D-Ala 部分被 D-Ala-D-Ser 取代，因此受到正向调节 一个两部分系统，VanRS。独特的是，万古霉素既不诱导也不高组成型表达 vanG 操纵子本身赋予艰难梭菌对万古霉素的抗性。尽管如此，许多临床和 实验室产生的耐万古霉素艰难梭菌菌株含有可增加 vanG 的 vanRS 突变 表达，强烈表明操纵子的高表达与其他突变一起起作用， 以利于抵抗力的发展。 我们发现，在缺乏艰难梭菌万古霉素敏感组氨酸激酶 VanS 的情况下， 另一种尚未经过基因鉴定并暂时命名为 KinX 的组氨酸激酶也对 万古霉素，能够取代 VanS 并诱导 vanG 操纵子。受调节的组氨酸激酶串扰 对相同环境信号（在本例中为万古霉素）的反应是不寻常的。与 VanS 相比，KinX 还 对至少一种干扰肽聚糖合成的抗生素有反应。因此，至关重要的是 详细了解 KinX 的功能非常重要，KinX 是根据临床使用的药物而激活的。 抗生素，可能通过 vanG 的调节导致艰难梭菌对万古霉素产生耐药性 操纵子，并且很可能调节参与肽聚糖代谢的其他基因。 使用几种独立、无偏见或有针对性的方法，包括 RNA-Seq 和 CRISPRi，我们 提议鉴定新型组氨酸激酶 KinX，以及可能控制其的同源反应调节因子 肽聚糖生物合成基因的表达。使用基因特异性和全局表达分析，我们 将确定 KinX 对 vanG 操纵子调节的贡献并定义 KinX 调节子。我们的 结果将为肽聚糖生物合成和万古霉素敏感性机制提供新的线索 艰难梭菌的耐药性。 万古霉素耐药菌株在临床上常见，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。 vanG监管详细知识 操纵子和肽聚糖代谢的其他基因对于理解肽聚糖代谢的发展至关重要 万古霉素耐药性和设计针对肽聚糖的新型抗菌药物。

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.