Role of YxdJK and DAK in the Enterococcal Envelope Stress Response

YxdJK 和 DAK 在肠球菌包膜应激反应中的作用

• 批准号: 10554055
• 负责人: William R Miller
• 金额: $ 17.12万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-03 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10554055
• 关键词: Role; YxdJK; DAK; Enterococcal; Envelope

Project Summary This K08 Career Development Award application is intended to support the acquisition of skills and knowledge needed to fulfill my long-term goal of becoming an independent physician-scientist focused on combating antimicrobial resistant organisms, a serious threat to medical practice worldwide. Vancomycin resistant enterococci (VRE) are an example of these pathogens and are a leading cause of healthcare associated infections affecting critically ill and immunocompromised patients. VRE are categorized by the CDC as a serious threat requiring the urgent development of novel therapeutic strategies. The lipopeptide antibiotic daptomycin (DAP) is now a front line agent for VRE infections, but resistance to DAP can arise while on therapy. The LiaFSR system, a major mediator of the cell envelope stress response, has been strongly implicated in the development of DAP resistance. Inactivation of this system by deletion of the gene encoding the LiaR response regulator was shown to re-sensitize enterococci to DAP. However, adaptation of LiaR deficient strains of both clinical and laboratory origin resulted in DAP resistance, suggesting that alternate pathways can protect the cell from antibiotic attack. Using whole genome sequencing of adapted strain pairs, I identified two pathways with novel contributions to DAP and cephalosporin resistance in enterococci, i) the YxdJK stress response system, and ii) the dihydroxyacetone kinase (DAK) domain protein involved in the metabolism of extracellular fatty acids. The YxdJK system consists of a sensor histidine kinase (YxdK), a DNA binding response regulator (YxdJ) and two ATP-binding cassette (ABC) transporters required to confer resistance to bacitracin. Deletion of the gene encoding the YxdJ response regulator sensitizes Enterococcus faecalis to both DAP and cephalosporins, despite a functional LiaFSR system. DAP-resistant strains using the above pathways appear to display a very distinct mechanism of resistance to cell-envelope acting antibiotics. This proposal is designed to dissect the role of the YxdJK system and the DAK enzyme in two major specific aims. First, I will characterize the contributions of the YxdJK system to the cell envelope stress response to antibiotics by defining how the system senses antibiotic stress and what genes are differentially expressed when the system is active. Second, I will determine the DAK mediated changes that alter membrane susceptibility to antibiotics, by comparing the membranes of wild type and DAK deletion strains to assess for changes in phospholipids, envelope structure, membrane protein function, and biofilm formation. The Center for Antimicrobial Resistance and Microbial Genomics (CARMiG) at the University of Texas Health Science Center and adjacent institutions of the Texas Medical Center will provide an unparalleled environment to grow as an investigator, with both an institutional commitment to combating antimicrobial resistance and an intensive mentorship program dedicated to helping junior faculty make the transition to independence.

项目摘要 该K08职业发展奖的申请旨在支持获取技能和知识 需要实现我成为一名独立医生 - 科学家的长期目标，专注于打击 抗菌抗菌生物，对全球医学实践的严重威胁。万古霉素耐药 肠球菌（VRE）是这些病原体的一个例子，是与医疗保健相关的主要原因 影响重症患者和免疫功能低下的患者的感染。 VRE由CDC分类为 严重威胁需要紧急发展新型治疗策略。脂蛋白抗生素 Daptomycin（DAP）现在是VRE感染的前线药物，但在启用时可能会出现对DAP的抗性 治疗。 LIAFSR系统是细胞包膜应力反应的主要介体，一直很强 与DAP抗性的发展有关。通过删除基因编码来灭活该系统 显示骗子响应调节器可将肠球菌重新敏感为DAP。但是，撒谎的适应 临床和实验室起源的不足菌株导致DAP抗性，表明交替 途径可以保护细胞免受抗生素攻击。使用适应应变对的整个基因组测序，I 鉴定出对肠球菌中DAP和头孢菌素耐药性的新颖贡献的两种途径，i） YXDJK应力响应系统和ii）二羟基丙酮激酶（DAK）域蛋白 细胞外脂肪酸的代谢。 YXDJK系统由传感器组氨酸激酶（YXDK）组成，DNA 结合响应调节器（YXDJ）和两个ATP结合盒（ABC）转运蛋白需要 对细菌蛋白的抗性。编码YXDJ响应调节器的基因删除敏感肠球菌 尽管有功能性的LIAFSR系统，但粪便群也可以dap和头孢菌素。使用DAP抗性菌株 上面的途径似乎表现出非常不同的对细胞 - 嵌入作用抗生素的抗性的机制。 该提案旨在剖析YxDJK系统和DAK酶在两个主要特异性方面的作用 目标。首先，我将表征YXDJK系统对细胞包膜应力响应的贡献 通过定义系统如何​​感觉抗生素应激和哪些基因差异表达来抗生素 当系统处于活动状态时。其次，我将确定改变膜的DAK介导的变化 通过比较野生型和DAK缺失菌株的膜来评估抗生素的敏感性 磷脂，包膜结构，膜蛋白功能和生物膜形成的变化。中心 德克萨斯大学健康科学的抗菌抗性和微生物基因组学（Carmig） 德克萨斯州医疗中心的中心和邻近机构将提供无与伦比的环境 作为研究者，既有对抗菌抗性的制度承诺，又有强度 致力于帮助初级教师过渡到独立的指导计划。