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

艰难梭菌肽聚糖合成中涉及的新型双组分系统的鉴定

• 批准号: 10624376
• 负责人: BORIS R BELITSKY
• 金额: $ 8.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-18 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10624376
• 关键词: Anabolism; Anaerobic Bacteria; Antibiotic Resistance; Antibiotics; Bacteria; Binding; CRISPR interference; Cell Wall; Cells; Clinic; Clinical; Clinical Research; Clostridium; Clostridium difficile; Development; Disease; Environment; Enzymes; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Human; Individual; Infection; Knowledge; Laboratories; Link; Lipids; Metabolic Pathway; Metabolism; Mutation; Names; Operon; Pathway interactions; Peptidoglycan; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Predisposition; Prokaryotic Cells; Pseudomembranous Colitis; Regulation; Regulatory Element; Regulon; Relaxation; Repression; Reproduction spores; Resistance; Resistance development; Role; Signal Pathway; Signal Transduction; Specificity; System; Vancomycin; Vancomycin Resistance; analog; antibiotic-associated diarrhea; antimicrobial; cell growth; constitutive expression; design; differential expression; emerging pathogen; mutant; novel; null mutation; pathogen; pathogenic bacteria; prevent; protein-histidine kinase; ramoplanin; resistant strain; response; transcriptome sequencing

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.

抽象的 梭状芽胞杆菌艰难梭菌是一种形成孢子的厌氧细菌，可能引起严重疾病，包括 人类中与抗生素相关的腹泻和假膜结肠炎。万古霉素是一线药物 治疗艰难梭菌感染；它靶向肽聚糖的生物合成，这是一种针对原核细胞和 细菌细胞壁和生长的形成至关重要。万古霉素与d-ala-d-ala残基结合 肽聚糖中间体的中间体，并防止其掺入成熟的肽聚糖中。 艰难梭菌vang操纵子，类似物的类似物在其他细菌中赋予万古霉素耐药性 由于用d-ala-d-ser替换了肽聚糖的D-Ala-d-ala部分，因此受到正面调节 两个组件系统VANRS。独特的是，万古霉素诱导的也不是高的构成表达 vang操纵子本身赋予了艰难梭菌中对万古霉素的抵抗。然而，许多临床和 实验室生成的万古霉素耐药性艰难梭菌菌株包含增加Vang的VANR突变 表达，强烈表明操纵子的高表达与其他突变一起贡献 抗药性的发展。 我们发现，在缺乏艰难梭菌万古霉素的组氨酸激酶，VANS，VANS， 另一个尚未鉴定出遗传鉴定和临时命名为kinx的组氨酸激酶也对 万古霉素能够取代货车并诱导vang操纵子。一个受调节的组氨酸激酶串扰 对同一环境信号的响应，在这种情况下，万古霉素是不寻常的。与货车相反，kinx也 对至少一种干扰肽聚糖合成的抗生素反应。因此，这是批判性的 重要的是要详细了解KINX的功能，该功能是根据临床使用的 抗生素可能通过调节Vang的调节导致艰难梭菌对万古霉素的新兴耐药性 操纵子，很可能会调节与肽聚糖代谢有关的其他基因。 使用几种独立的公正或有针对性的方法，包括RNA-Seq和Crispri，我们 建议识别新型的组氨酸激酶，kinx，及其可能控制的同源响应调节剂 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 Operon调节的贡献，并定义Kinx调节。我们的 结果将为肽聚糖生物合成和万古霉素敏感性的机制提供新的启示 艰难梭菌的阻力。 在诊所中通常检测到万古霉素抗性菌株，耐药性的传播可能 成为治疗艰难梭菌感染的严重问题。关于调节的详细知识 肽聚糖代谢的操纵子和其他基因对于理解的发展至关重要 万古霉素的耐药性和设计针对肽聚糖的新抗菌剂。