Characterizing a Self-Digesting-Mediated Reversible Drug Tolerance Mechanism in Bacteria

摘要 通过将我们在永久细胞生物学方面的专业知识与先进的当前技术相结合，我们在这方面的总体目标是 项目是描述细菌中一种自我消化介导的持久性机制，并探索 这一过程的治疗潜力。细菌持续体是一种罕见的暂时耐受的表型变异体 到高浓度的抗生素。这些变异通常是遗传上相同的非生长细胞。 他们对抗生素敏感的亲属。持久细胞促进慢性感染的复发，并作为 蓄水池中出现了耐药突变株。因此，消除这些细胞可以改善临床 大多数医院治疗的感染的结果，但消除持久性感染的有效方法仍然存在 有限的。这一提议的中心假设是自我消化是宿存细胞形成的一种机制。 在细菌物种中。因此，破译这一机制的基本组成部分可能会提供 一种全球治疗方法，因为自我消化是许多细菌物种的标志。在我们之前的研究中， 我们发现，持续体大多来自具有高氧化还原活性的静止相细胞，即 由内源蛋白质和RNA降解(即自我消化)维持。我们进一步确定了这一损失 静止期代谢活动的减少通过阻止内源性消化来降低持续期水平 蛋白质和RNA，产生对抗生素敏感性增强的细胞。在这些充满希望的结果的鼓舞下，我们 提出以下具体目标，以探索我们的中心假设。(目标1)我们将绘制自我消化- 我们的模式生物，大肠杆菌，使用荧光激活细胞分选的相关机制，记者 质粒，基因缺失，化学抑制剂，代谢组学技术，以及我们拥有的新的分析方法 开发的目的是量化持久性细胞、可存活但不可培养的细胞和细胞内降解。我们将进一步 使用一种与临床相关的微生物铜绿假单胞菌来验证我们的假设，这是 免疫系统受损的囊性纤维化患者发病和死亡的主要原因。(目标 2)我们将利用一种可降解的荧光蛋白来开发一种新的筛选方法来快速鉴定 通过干扰大肠杆菌中的自我消化机制来消除蠕动细胞的化合物 和铜绿假单胞菌。候选抑制剂对持久性水平的影响将在体内进一步测试 在高细胞密度感染的小鼠模型中的条件。我们的研究在许多层面上都是新颖和有意义的。 我们解决核心假设的方法在概念上是创新的。此外，这一映射 全面的细菌途径，从最初的外源触发，到信号转导，再到源头 抗生素耐受性的提高，将使我们能够开发出有效的抗耐药药物。最后，本研究 该计划将通过提供一个平台来研究不同细菌物种和 作为实验室研究和临床试验之间的桥梁。

SUMMARY By integrating our expertise in persister cell biology with advanced current technologies, our overall goals in this project are to characterize a self-digestion-mediated persistence mechanism in bacteria and to explore the therapeutic potential of this process. Bacterial persisters are rare phenotypic variants that are temporarily tolerant to high concentrations of antibiotics. These variants are generally nongrowing cells that are genetically identical to their antibiotic-susceptible kin. Persister cells facilitate the recurrence of chronic infections and serve as a reservoir for the emergence of drug resistance mutants. As such, elimination of these cells improves clinical outcomes for the majority of hospital-treated infections, but effective methods for persister elimination remain limited. The central hypothesis of this proposal is that self-digestion is a mechanism for persister cell formation in bacterial species. Therefore, deciphering the essential components of this mechanism can potentially provide a global treatment approach, as self-digestion is a hallmark of many bacterial species. In our previous studies, we discovered that persisters are mostly derived from stationary-phase cells with a high redox activity that is maintained by endogenous protein and RNA degradation (i.e., self-digestion). We further determined that loss of stationary-phase metabolic activity reduces persister levels by preventing the digestion of endogenous proteins and RNA, yielding cells with enhanced antibiotic sensitivity. Inspired by these promising results, we propose the following specific aims to explore our central hypothesis. (Aim 1) We will map the self-digestion- related mechanisms in our model organism, Escherichia coli, using fluorescence-activated cell sorting, reporter plasmids, gene deletions, chemical inhibitors, metabolomics technology, and novel assays that we have developed to quantify persisters, viable but non-culturable cells, and intracellular degradation. We will further test our hypothesis using a clinically relevant microorganism, Pseudomonas aeruginosa, which is the predominant cause of morbidity and mortality in cystic fibrosis patients with compromised immune systems. (Aim 2) We will utilize a degradable fluorescent protein to develop a novel screening approach for rapidly identifying chemical compounds that can eradicate persister cells by perturbing the self-digestion mechanisms in E. coli and P. aeruginosa. The effects of candidate inhibitors on persister levels will be further tested under in vivo conditions in a mouse model of high cell density infections. Our study is novel and significant on many levels. Our approach to address our central hypothesis is conceptually innovative. In addition, mapping of this comprehensive bacterial pathway from its initial exogenous trigger, through its signal transduction, to the source of antibiotic tolerance, will enable us to develop affective antipersister therapeutics. Finally, this research program will have a clinical impact by providing a platform to study persistence in different bacterial species and by serving as a bridge from laboratory investigations to clinical trials.