Role of YxdJK and DAK in the Enterococcal Envelope Stress Response

项目摘要 此K08职业发展奖申请旨在支持获得技能和知识 我需要实现我的长期目标，成为一名专注于对抗的独立内科科学家 具有抗菌素耐药性的生物，严重威胁着全球的医疗实践。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 影响危重病人和免疫功能低下病人的感染。VRE被疾病预防控制中心归类为 严重的威胁要求迫切开发新的治疗策略。脂肽类抗生素 达托霉素(DAP)现在是治疗VRE感染的一线药物，但在服药期间可能会产生抗药性 心理治疗。作为细胞被膜应激反应的主要中介，LianFSR系统一直是强烈的 与DAP耐药的发生有关。编码基因的缺失使这一系统失活 说谎者反应调节剂被证明能使肠球菌对DAP重新敏感。然而，《说谎者》的改编 临床和实验室来源的缺陷菌株导致对DAP的耐药性，这表明 途径可以保护细胞免受抗生素的攻击。利用适应菌株对的全基因组测序，我 确定了两条对肠球菌DAP和头孢菌素耐药有新贡献的途径，i) YxdJK应激反应系统，以及ii)二羟丙酮激酶(DAK)结构域蛋白参与 胞外脂肪酸的代谢。YxdJK系统由传感器组氨酸激酶(YxdK)、DNA 结合反应调节子(YxdJ)和两个ATP结合盒(ABC)转运体需要授予 对杆菌肽的抗药性。YxdJ反应调节基因缺失使肠球菌增敏 粪便对DAP和头孢菌素类药物都有作用，尽管LianFSR系统起作用。使用DAP抗药性菌株 上述途径似乎表现出对细胞膜作用抗生素的非常独特的耐药机制。 这项建议旨在剖析YxdJK系统和DAK酶在两个主要特定的 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知抗生素压力以及哪些基因有差异表达来定义抗生素 当系统处于活动状态时。其次，我将确定DAK介导的改变膜的变化 通过比较野生型和DAK缺失菌株的细胞膜来评估对抗生素的敏感性 磷脂、包膜结构、膜蛋白功能和生物膜形成的变化。《中心》 得克萨斯大学的抗菌素耐药性和微生物基因组学(CARMiG) 德克萨斯医疗中心及其邻近机构将提供无与伦比的发展环境 作为一名调查员，既有对抗抗菌素耐药性的机构承诺，又有密集的 导师计划致力于帮助初级教员过渡到独立。

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.