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伤的恢复能力，包括缺乏有效治疗的慢性和复发性细菌感染。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并阻止清除，以及宿主如何避免对病原体存在的有害炎症反应。在我们之前的工作中，我们发现了一种由铜绿假单胞菌（PA）分泌的小挥发性QS分子，2-氨基苯乙酮（2-AA），它作为一种跨界的“信息化学”，以改变免疫反应和代谢，从而训练宿主增加宿主耐受性/弹性（HT/R），这被定义为宿主应对细菌遭遇而不随之降低适应性的能力；这些改变使病原体避免被消灭并在哺乳动物组织中持续存在。本申请的具体目标是以范例的方式破译细菌产物对宿主代谢组和表观基因组的作用模式，以便深入了解QS分子重编程宿主代谢组和表观基因组以促进HT/R的第一个机制例子。我们的假设是，2-AA通过组蛋白去乙酰化酶1 （HDAC1）的持续活性施加的长期免疫调节变化源于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确定HT/R的细菌-宿主相互适应度。我们建议通过使用PA的实验来实现我们的目标，PA是一种顽固性革兰氏阴性ESKAPE细菌，可以抵抗抗生素的根除，形成生物膜，并举例说明当前临床有问题的病原体。在Aim 1中，我们将通过评估2-AA耐受促进体内免疫记忆的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Aim 2中，我们将探讨2- aa调节组织中巨噬细胞自噬的能力，并对关键宿主和允许细菌持续存在的细胞机制进行细致的分析。在Aim 3中，除了人原代巨噬细胞外，我们还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改变之间的相互作用。这些变化对细菌持久性、组蛋白乙酰化、能量代谢和我们的新型抗2aa化合物的功效的影响将通过临床相关的感染动物模型进行评估。在Aim 4中，我们将寻求使用点击化学来确定2-AA的直接目标。提出的“宿主耐受训练”机制的阐明旨在提高我们对仍然非常有限的HT/R的理解。在这种情况下，这些发现可能为开发治疗性治疗提供了机会，这些治疗性治疗可以训练宿主增强对感染的抵抗力。原则上，由于QS在革兰氏阴性和革兰氏阳性的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，因此所获得的知识也可适用于其他细菌引发的持续性感染。

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.