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技能和知识的获取 我需要实现我的长期目标，成为一名独立的物理学家，科学家，专注于打击 抗生素耐药性微生物，对全球医疗实践构成严重威胁。万古霉素耐药 肠球菌（VRE）是这些病原体的一个例子，并且是与健康护理相关的疾病的主要原因。 影响重症和免疫功能低下患者的感染。VRE被CDC归类为 严重的威胁，迫切需要开发新的治疗策略。脂肽类抗生素 达托霉素（DAP）现在是VRE感染的一线药物，但对DAP的耐药性可能会在治疗期间出现。 疗法LiaFSR系统是细胞被膜应激反应的主要介质， 与DAP耐药性的发展有关。通过删除编码 LiaR反应调节剂显示使肠球菌对DAP再敏感。然而，LiaR的改编 临床和实验室来源的缺陷菌株导致DAP抗性，这表明替代的 可以保护细胞免受抗生素的攻击。使用适应菌株对的全基因组测序， 确定了两种途径，对肠球菌中的DAP和头孢菌素耐药性有新的贡献，i） YxdJK应激反应系统，和ii）参与该系统的二羟丙酮激酶（DAK）结构域蛋白， 细胞外脂肪酸的代谢。YxdJK系统由传感器组氨酸激酶（YxdK）、DNA聚合酶（DNA聚合酶）和DNA聚合酶组成。 结合反应调节因子（YxdJ）和两个ATP结合盒（ABC）转运蛋白， 对杆菌肽的耐药性。编码YxdJ反应调节因子的基因缺失使肠球菌致敏 粪肠球菌的DAP和头孢菌素，尽管功能LiaFSR系统。DAP耐药菌株使用 上述途径似乎显示出对细胞包膜作用抗生素的非常独特的抗性机制。 该建议旨在剖析YxdJK系统和DAK酶在两种主要的特异性免疫应答中的作用。 目标。首先，我将描述YxdJK系统对细胞包膜应激反应的贡献， 通过定义系统如何感知抗生素应激以及哪些基因差异表达， 当系统激活时。其次，我将确定DAK介导的改变， 对抗生素的敏感性，通过比较野生型和DAK缺失菌株的膜来评估 磷脂、包膜结构、膜蛋白功能和生物膜形成的变化。中心 抗微生物耐药性和微生物基因组学（CARMiG）在得克萨斯大学健康科学 中心和德克萨斯州医疗中心的相邻机构将提供无与伦比的成长环境 作为一名研究者，既有对抗抗生素耐药性的机构承诺， 导师计划致力于帮助初级教师过渡到独立。

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.