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

摘要 艰难梭菌是一种能形成孢子的厌氧细菌，可引起严重疾病，包括 人类腹泻和伪膜性结肠炎。万古霉素是一线药物 治疗C.艰难梭菌感染;它靶向肽聚糖生物合成，这是一种对原核细胞特异性的途径， 对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 并阻止其掺入成熟肽聚糖。 梭艰难梭菌旺操纵子，其类似物在其他细菌物种中赋予万古霉素抗性 由于肽聚糖的D-Ala-D-Ala部分被D-Ala-D-Ser取代， 双组分系统VanRS。独特的是，万古霉素既不诱导也不高，组成型表达， 旺操纵子本身赋予C.很难尽管如此，许多临床和 实验室产生万古霉素抗性C.艰难梭菌菌株含有vanRS突变， 表达，强烈表明操纵子的高表达与其他突变一起， 抵抗运动的发展 我们发现，在C.艰难梭菌万古霉素敏感组氨酸激酶，VanS， 另一种组氨酸激酶，尚未遗传鉴定，暂时命名为KinX，也响应于 万古霉素，并能够取代VanS和诱导旺操纵子。一种受调节的组氨酸激酶串扰， 对相同环境信号的反应，在这种情况下万古霉素是不寻常的。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， 重要的是要详细了解KinX的功能，它是响应于临床使用的 抗生素，可能有助于C.通过调节旺， 操纵子，并且很可能调节参与肽聚糖代谢的其他基因。 使用几种独立的无偏或靶向方法，包括RNA-Seq和CRISPRi，我们 我建议确定新的组氨酸激酶，KinX，并可能，其同源反应调节器，控制 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对旺操纵子的调节的贡献并定义KinX调节子。我们 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， C.抗性很难 万古霉素耐药菌株在临床上是常见的，耐药的传播可能会导致耐药菌株的感染。 成为治疗C.艰难感染关于旺调节的详细知识 操纵子和肽聚糖代谢的其他基因对于理解 万古霉素耐药性和设计靶向肽聚糖的新抗菌剂。

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.