Molecular and Metabolic inter-kingdom actions of a bacterial quorum sensing signal in promotion of host tolerance/resilience.

我们的长期目标是开发治疗方法，以提高严重感染患者对病原体损害的韧性，包括慢性和复发性细菌感染，而这些感染缺乏有效的治疗方法。细菌群体感应(QS)信号是免疫调节的重要介质。然而，目前尚不清楚微生物如何利用免疫调节信号来维持长期存在和阻碍清除，以及宿主如何避免对病原体存在的有害炎症反应。在我们以前的工作中，我们发现了铜绿假单胞菌(PA)分泌的一个小的挥发性QS分子，2-氨基苯乙酮(2-AA)，它作为一种跨王国的信息化学物质，以训练宿主增加宿主耐受性/弹性(HT/R)的方式改变免疫反应和新陈代谢，这被定义为宿主应对细菌接触的能力，而不会因此降低适合度；这些变化使病原体能够避免消除并持续存在于哺乳动物组织中。这项应用的具体目标是典型地破译细菌产物对宿主代谢组和表观基因组的作用模式，以获得对QS分子的第一个机制示例的洞察，QS分子重新编程宿主代谢组和表观基因组以促进HT/R。我们的假设是，2-AA通过组蛋白脱乙酰基酶1(HDAC1)的持续活性而施加的长期免疫调节变化来自2-AA促进的代谢改变；宿主代谢组和表观基因组之间的相互作用有助于定义HT/R的相互细菌-宿主适合性。我们建议通过使用PA的实验来实现我们的目标，PA是一种顽固的革兰氏阴性ESKAPE细菌，它无法被抗生素根除，形成生物膜，并例证了目前临床上存在问题的病原体。在目标1中，我们将通过评估2-AA耐受促进体内免疫记忆中长期免疫调节的分子机制来研究2-AA如何维持表观遗传重编程，并通过功能研究确定发生的代谢变化。在目标2中，我们将探讨2-AA调节组织中巨噬细胞自噬的能力，并对允许细菌持续存在的关键宿主角色和细胞机制进行细致的分析。在目标3中，除了人的原代巨噬细胞外，我们还将使用小鼠体外和体内系统，通过分子和功能方法评估2-AA促进的表观遗传控制和代谢变化之间的相互作用。这些变化对细菌总数、组蛋白乙酰化、能量代谢的影响以及我们新的抗2AA化合物的有效性将使用临床相关的感染动物模型进行评估。在目标4中，我们将利用点击化学来寻找2-AA的直接靶标。提出的“宿主耐受训练”机制的解释是为了提高我们对仍然极其有限的HT/R的理解。在这种情况下，这些发现可能会为应用于治疗疗法的开发提供机会，这些疗法可以训练宿主增强对感染的抵抗力。原则上，所获得的知识可能适用于也由其他细菌引发的持续性感染，因为QS在革兰氏阴性和革兰氏阳性人类细菌病原体中广泛保守，因此，2-AA样分子可能存在于其他细菌中。

Our long-term goal is to develop treatments that may improve resilience to pathogen damage in patients with serious infections, including chronic and relapsing bacterial infections, for which effective treatments are lacking. Bacterial quorum sensing (QS) signals are important mediators of immunomodulation. However, it remains unclear how microbes utilize immunomodulatory signals to maintain a long-term presence and thwart clearance and how hosts avoid harmful inflammatory response to a pathogen’s presence. In our previous work, we identified a small volatile QS molecule excreted by Pseudomonas aeruginosa (PA), 2-aminoacetophenone (2-AA), that acts as an inter-kingdom “infochemical” to alter immune responses and metabolism in a manner that trains the host to increase host tolerance/resilience (HT/R), which is defined as the host's ability to cope with bacterial encounter without a consequent reduction in fitness; these alterations allow the pathogen to avoid elimination and persist in mammalian tissues. The specific goal of this application is to decipher paradigmatically the mode of action of a bacterial product on host metabolome and epigenome in order to gain insights into the first mechanistic example of a QS molecule that reprograms the host metabolome and epigenome to promote HT/R. Our hypothesis is that the long-lasting immunomodulatory changes exerted by 2-AA through the sustained activity of histone deacetylase 1 (HDAC1) derive from 2-AA–promoted metabolic alterations; and that the interplay between host metabolome and epigenome contribute to the mutual bacterial-host fitness that defines HT/R. We propose to achieve our goal through experiments employing PA, a recalcitrant Gram-negative ESKAPE bacterium that defies eradication by antibiotics, forms biofilms, and exemplifies current clinically problematic pathogens. In Aim 1, we will examine how 2-AA maintains epigenetic reprogramming by assessing the molecular mechanisms of long-term immunomodulation in immune memory in vivo promoted by 2-AA tolerization, and determine the occurring metabolic changes by performing functional studies. In Aim 2, we will interogate the capacity of 2-AA to regulate macrophage autophagy in tissues and conduct a meticulous analysis of the key host players and cellular mechanisms that permit bacterial persistence. In Aim 3, using murine in vitro and in vivo systems in addition to human primary macrophages, we will evaluate the interplay between 2-AA promoted epigenetic control and metabolic alterations by undertaking molecular and functional approaches. The impact of these alterations in bacterial persitence, histone acetylation, energy metabolism and the efficacy of our novel anti-2AA compound will be asssessed using clinically relevant animal models of infection. In Aim 4, we will seek to identify 2-AA’s direct target using click chemistry. The proposed elucidation of “host tolerance training” mechanisms is intended to improve our understanding of HT/R that remains extremely limited. In this context, the findings may offer opportunities for being applied to the development of therapeutic treatments that can train the host to augment resilience to infection. In principle, the knowledge to be obtained could be applicable to persistent infections triggered also by other bacteria because QS is broadly conserved among Gram-negative and Gram-positive human bacterial pathogens and, as such, 2-AA-like molecules are likely to exist in other bacteria.