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，也是对公共卫生的主要威胁 由于耐多药(MDR)菌株的持续上升。因为这种病毒的致病潜能 肠球菌与其无处不在的应激韧性密切相关，我们实验室的工作旨在识别和解剖 允许粪肠球菌，人类感染中最常见的肠球菌种， 在充满敌意的宿主环境中生存。第二信使核苷酸，如(P)ppGpp(效应器 严格反应的分子)和c-diamp是由细菌对内部或外部的反应而产生的 刺激，在调节与细胞内稳态和毒力相关的过程中发挥主要作用。在……里面 此外，环核苷酸如c-diamp通过以下途径作为哺乳动物先天免疫反应的激动剂 刺激强烈的刺痛依赖型I型干扰素反应。有趣的是，之前的调查 表明c-di-AMP和(P)ppGpp信号网络在其他细菌中是相互连接的，但明显 对这种相互作用的机制和生理意义仍然缺乏了解。在以前的 研究发现，粪肠球菌依靠少量的(P)ppGpp维持平衡。 并能准确、及时地对外界提示做出反应。我们还发现，缺乏 (P)ppGpp大大提高了对抗生素的敏感性，并减弱了粪肠球菌的毒力。最近，我们 表明c-diamp对粪肠球菌的病理生理学也是至关重要的，无论c-diamp的积聚或缺乏。 Di-AMP可显著削弱粪肠球菌的毒力潜力。本申请的目标是：(I) 利用导尿管相关尿路探讨c-二AMP在感染过程中的多方面作用 感染(CAUTI)小鼠模型，概括了人类疾病的许多临床特征， (Ii)鉴定和鉴定粪肠球菌中的c-diamp结合/效应蛋白；及。(Iii)研究 C-di-AMP和(P)ppGpp监管网络的整合如何控制促进 细菌适合性，然后确定这种联系如何有助于粪肠球菌的病理生理学。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 我们的研究团队在这一应用程序的每个特定领域拥有专业知识和可靠的跟踪记录。的重要意义 拟议的研究在于揭示c-di-AMP在宿主-病原体相互作用中的多方面作用， 揭示了c-di-AMP在主要MDR病原体中的调控范围，并揭示了 C-di-AMP和(P)ppGpp之间的复杂关系。鉴于调控核苷酸在人类基因组中的核心作用 细菌的发病机制，更好地了解它们如何在鉴定的基础上调节细胞生理 而对它们的作用机制和效应器的表征可以帮助合理设计新的 抗菌疗法。

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 signaling 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 regulatory 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. Given the central role of regulatory nucleotides in bacterial pathogenesis, a better understanding of how they modulate cell physiology based on identification and characterization of their mechanisms of action and effectors can facilitate the rational design of new antimicrobial therapies.