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

总结 通过将我们在持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： 项目是表征细菌中自我消化介导的持久性机制，并探索 这个过程的治疗潜力。细菌持久性是罕见的表型变异，暂时耐受 高浓度的抗生素。这些变异体通常是基因相同的非生长细胞 给他们的易感亲戚持续存在的细胞促进慢性感染的复发， 耐药性突变体的出现。因此，消除这些细胞改善了临床 大多数医院治疗的感染的结果，但有效的方法，持久消除仍然存在 有限公司这个提议的中心假设是自我消化是持续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以潜在地提供 这是一种全球性的治疗方法，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，persisters主要来自静止期细胞，具有高氧化还原活性， 通过内源性蛋白质和RNA降解来维持（即，自我消化）。我们进一步确定， 静止期代谢活动的减少通过阻止内源性代谢物的消化来降低持久水平。 蛋白质和RNA，产生具有增强的抗生素敏感性的细胞。受这些有希望的结果的启发，我们 提出以下具体目标来探讨我们的中心假设。(Aim 1）我们将绘制自我消化图- 在我们的模式生物，大肠杆菌，使用荧光激活细胞分选，报告相关机制 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检测方法， 开发用于定量持久性，可存活但不可培养的细胞和细胞内降解。我们将进一步 使用临床相关微生物铜绿假单胞菌来验证我们的假设， 免疫系统受损的囊性纤维化患者发病率和死亡率的主要原因。（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， 通过干扰大肠杆菌的自我消化机制来根除持留细胞的化合物。杆菌 和铜绿假单胞菌。候选抑制剂对持久水平的影响将在体内试验中进一步测试。 在高细胞密度感染的小鼠模型中，我们的研究在许多层面上都是新颖的和有意义的。 我们解决中心假设的方法在概念上是创新的。此外，映射此 全面的细菌途径，从其最初的外源性触发，通过其信号转导， 抗生素耐药性的研究，将使我们能够开发有效的抗持续性治疗方法。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的持久性， 作为从实验室研究到临床试验的桥梁。

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.