Mechanistic basis of how LD-transpeptidases protect against outer membrane defects

项目摘要/摘要 多重药物和广泛耐药的革兰氏阴性细菌的出现是一个日益严重的问题， 威胁到既定的抗菌治疗方案。鲍曼不动杆菌是一种严重威胁致病菌 臭名昭著的是它迅速产生多药耐药性的能力。鲍曼不动杆菌会引起医院获得性感染， 主要表现为菌血症、尿路和伤口感染。在美国，估计有60%的医院获得 鲍曼不动杆菌感染是多重耐药的，通常包括碳青霉烯类耐药，这使得粘菌素成为 “最后手段”的治疗方案。然而，粘菌素耐药性也出现了。有迫切的需要 了解鲍曼不动杆菌耐药的内在机制以指导替代抗菌药物 战略。我们的初步工作已经确定了两种促进粘菌素抗性活性的LD-转肽酶 脂寡糖缺陷型鲍曼不动杆菌。具体地说，依赖LD转肽酶的细胞包膜修饰 是抗性表型的关键，在抗性表型中，替代交联剂弥补外膜缺陷。在这 提议，我们将解决三个重要问题，以了解LD-转肽酶的功能和调节 在鲍曼不动杆菌中，包括(I)LD-转肽酶活性如何对抗其他溶解的机械力 (Ii)鲍曼不动杆菌的LdtK脂蛋白底物是如何调节的？(Iii) A类青霉素结合蛋白如何影响鲍曼不动杆菌的转肽酶活性？总而言之，这些 研究将解决细菌生理学和细胞膜组装的关键问题，这将使我们能够 建立鲍曼不动杆菌多药耐药的内在因素模型。此外，这些分析 将帮助设计针对基本外膜和肽聚糖的组合药物方案 因此，我们的发现支持国家 健康研究所的使命，旨在促进减少人类疾病的基本发现。

Project Summary/Abstract The emergence of multidrug and extensively drug-resistant Gram-negative bacteria is a growing problem that threatens established antimicrobial treatment protocols. Acinetobacter baumannii is a critical threat pathogen notorious for its ability to rapidly develop multidrug resistance. A. baumannii causes hospital-acquired infections, which manifest as bacteremia, urinary tract and wound infections. In the US, an estimated 60% of hospital-acquired A. baumannii infections were multidrug-resistant, often including carbapenem resistance, which leaves colistin as the “last-resort” treatment option. However, colistin resistance has also emerged. There is an urgent need to understand intrinsic mechanisms that promote antibiotic resistance in A. baumannii to guide alternative antimicrobial strategies. Our preliminary work has identified two LD-transpeptidases that promote viability of colistin resistant lipooligosaccharide-deficient A. baumannii. Specifically, LD-transpeptidase-dependent cell envelope modifications are key for the resistance phenotype, where alternative crosslinks compensate for outer membrane defects. In this proposal, we will address three important questions to understand the function and regulation of LD-transpeptidases in A. baumannii, including (I) how does LD-transpeptidase activity counter otherwise lytic mechanical forces produced by outer membrane defects?; (II) how are LdtK lipoprotein substrates regulated in A. baumannii?; and (III) how do class A penicillin-binding proteins impact LD-transpeptidase activity in A. baumannii? Collectively, these studies will address key questions in bacterial physiology and cell envelope assembly, which will enable us to build a model of intrinsic factors in A. baumannii that contribute to multidrug resistance. Furthermore, these analyses will aid in the design of combinatorial drug regimens that target both essential outer membrane and peptidoglycan layers of the cell envelope, thus precluding resistance; consequently, our findings support the National Institute of Health mission, which aims to foster fundamental discoveries to reduce human disease.