Second Messenger Nucleotides of Enterococcus faecalis

摘要 肠球菌是医院获得性感染的第三大常见原因，也是一个主要的公共卫生威胁 由于多药耐药（MDR）菌株的不断增加。因为潜在的致病性 肠球菌与它们无处不在的压力恢复能力密切相关，我们实验室的工作旨在识别和分析 粪肠球菌（Enterococcus faecalis）是人类感染中最常见的肠球菌， 在恶劣的环境中生存。第二信使核苷酸如（p）ppGpp（效应子 严格反应的分子）和c-di-AMP由细菌响应内部或外部环境而产生。 刺激，在调节与细胞稳态和毒力相关的过程中发挥主要作用。在 此外，环核苷酸如c-di-AMP通过以下方式作为人的先天性免疫应答的激动剂： 刺激有效的STING依赖性I型干扰素应答。有趣的是，以前的调查 表明c-di-AMP和（p）ppGpp网络在其他细菌中相互连接，但清楚地表明， 对这种相互作用的机制和生理意义的理解仍然缺乏。前几 研究发现，E.粪便依赖于少量的（p）ppGpp来维持平衡 以准确和及时的方式对外部线索作出反应。我们还发现，缺乏 （p）ppGpp显著提高了E.粪便最近，我们 表明c-di-AMP对E.粪便病理生理学，无论是积累或缺乏c- di-AMP能显著削弱E.粪便本申请的目的是：（i） 探讨c-di-AMP在使用导管相关尿路感染期间所起的多方面作用 感染（CITTI）小鼠模型，其再现了人类中该疾病的许多临床特征， (ii)鉴定和表征大肠杆菌中的c-di-AMP结合/效应蛋白。粪便，以及（iii）调查 c-di-AMP和（p）ppGpp网络的整合如何控制促进细菌生长的特定过程 适应性，然后确定这种关联如何有助于E。粪便病理生理学这在概念上 创新的应用建立在我们广泛的初步数据和互补的专业知识和坚实的基础上， 我们的研究团队在此应用程序的每个特定领域的跟踪记录。建议的意义 研究的重点在于揭示c-di-AMP在宿主-病原体相互作用中的多方面作用， c-di-AMP在主要MDR病原体中的调节范围，并为复杂的关系提供了新的见解 在c-di-AMP和（p）ppGpp信令网络之间。考虑到第二信使核苷酸在 细菌的发病机制，更好地了解他们如何调节细胞生理学的基础上的鉴定 其作用机制和效应器的表征可以促进新的药物的合理设计。 抗菌治疗

ABSTRACT Enterococci are the 3rd most common cause of hospital-acquired infections and a major public health threat due to the continuous rise of multidrug-resistant (MDR) isolates. Because the pathogenic potential of enterococci is closely linked to their ubiquitous stress resilience, work in our lab aims to identify and dissect the mechanisms that allow Enterococcus faecalis, the most prevalent enterococcal species of human infections, to survive in the hostile host environment. Second messenger nucleotides such as (p)ppGpp (the effector molecule of the stringent response) and c-di-AMP are produced by bacteria in response to internal or external stimuli, playing major roles in the regulation of processes associated with cell homeostasis and virulence. In addition, cyclic nucleotides such as c-di-AMP act as agonists of the innate immune response of mammalians by stimulating a potent STING-dependent type I interferon response. Of interest, previous investigations have shown that the c-di-AMP and (p)ppGpp networks are interconnected in other bacteria, but a clear understanding of the mechanics and physiological significance of this interaction are still lacking. In previous studies, we discovered that E. faecalis depends on small amounts of (p)ppGpp to maintain a balanced metabolism and to respond to external cues in an accurate and timely manner. We also found that lack of (p)ppGpp greatly increases antibiotic sensitivity and attenuates the virulence of E. faecalis. More recently, we showed that c-di-AMP is also critical to E. faecalis pathophysiology and that either accumulation or lack of c- di-AMP can drastically impair the virulence potential of E. faecalis. The goals of this application are: (i) to probe the multifaceted roles played by c-di-AMP during infection using a catheter-associated urinary tract infection (CAUTI) mouse model that recapitulates many of the clinical characteristics of the disease in humans, (ii) to identify and characterize the c-di-AMP binding/effector proteins in E. faecalis, and (iii) to investigate how integration of the c-di-AMP and (p)ppGpp networks controls specific processes that promote bacterial fitness and then determine how this association contributes to E. faecalis pathophysiology. This conceptually innovative application builds on our extensive preliminary data and the complementary expertise and solid track record of our research team in each specific area of this application. Significance of the proposed studies lies in unravelling the multifaceted role of c-di-AMP in host-pathogen interactions, uncovering the scope of c-di-AMP regulation in a major MDR pathogen, and shedding new light onto the intricate relationship between c-di-AMP and (p)ppGpp signaling networks. Given the central role of second messenger nucleotides in bacterial pathogenesis, a better understanding of how they modulate cell physiology based on the identification and characterization of their mechanisms of action and effectors can facilitate the rational design of new antimicrobial therapies.