Investigating metabolism and DNA damage repair in uropathogenic Escherichia coli fluoroquinolone persisters

项目总结： 尿路致病性大肠杆菌(UPEC)是尿路感染(UTIs)的主要病原体之一。 随着抗生素治疗失败的频率越来越高，抗生素发现的速度越来越慢，复发率 尿路感染将继续增加。尽管抗药性病原体特别令人担忧， 治疗失败往往是由非遗传机制造成的，没有检测到耐药性。其中一个非- 抗生素治疗失败的遗传机制-持久性-以抗生素耐受性为特征 易感人群中的一小群细胞。细菌持久者，它可以可逆地退出抗生素- 药物撤除后的耐受状态，能够重新感染并阻碍治疗的成功。 众所周知，持久者在生长缓慢的种群中得到丰富，在这种种群中，总体细胞活动和新陈代谢 减少；然而，缺乏关于周围营养环境如何影响的知识 细菌生理学，以及这些因素如何影响对抗生素的敏感性。 我们的中心目标是确定碳源可用性对UPEC持久性的影响 氟喹诺酮类(FQ)抗生素，通过靶向DNA拓扑异构酶和 DNA损伤的累积。目标1旨在解决新陈代谢、生物分子合成和DNA的变化 在FQ处理期间响应碳源可用性的完整性。我们将通过以下方式评估这些因素 使用基于荧光的探针进行实验，以及代谢物的质谱分析。 这些数据将阐明碳源的可获得性如何影响细胞对 FQS，使我们能够探索作为增强靶点的特定机制。目标2将使我们了解如何 FQ处理后，DNA损伤反应的协调性因碳源的可获得性而发生变化。我们 将在大肠杆菌中部署强大的遗传方法和RNA测序来询问基因的时间 表达和DNA修复。这些目标的完成将使人们对各种DNA的重要性有更深的了解 FQ治疗后的修复机制。 总体而言，这些目标的完成将揭示潜在的代谢、生物合成和表达变化 FQ在大肠杆菌中的持久性。这一信息可以阐明潜在的基因，这些基因可以作为靶点来增强 FQS的活性和减少外周后代的抗性发展。此外，这些发现可能会导致 对临床感染的有趣暗示，宿主新陈代谢和 抗生素的功效。这项研究可能会突出添加葡萄糖以及其他代谢物的潜力，如 在抗生素给药期间添加化合物，以改善感染的治疗。

PROJECT SUMMARY: Uropathogenic Escherichia coli (UPEC) is one of the major causative agents of urinary tract infections (UTIs). With growing frequency of antibiotic treatment failure and slowing of antibiotic discovery, the rate of recurrent UTIs is poised to continue to increase. Although antibiotic resistant pathogens are of particular concern, treatment failure can often occur by non-genetic mechanisms, without detectable resistance. One of the non- genetic mechanisms of antibiotic treatment failure—persistence—is characterized by antibiotic tolerance in a small group of cells among a susceptible population. Bacterial persisters, which can reversibly exit the antibiotic- tolerant state following drug removal, is able to repopulate an infection and impede success of treatment. Persisters are known to be enriched in slow growing populations, where overall cellular activity and metabolism is reduced; however, there is a lack of knowledge on how the surrounding nutrient environment can affect bacterial physiology, and how these factors affect susceptibility to antibiotics. Our central objective is to determine the impact of carbon source availability on UPEC persistence to fluoroquinolone (FQ) antibiotics, which are bactericidal through the targeting of DNA topoisomerases and the accumulation of DNA damage. Aim 1 aims to address changes to metabolism, biomolecular synthesis, and DNA integrity in response to carbon source availability during FQ treatment. We will assess these factors by performing experiments with fluorescence-based probes, as well as mass spectrometry analysis of metabolites. These data will elucidate the ways by which carbon source availability can impact the susceptibility of cells to FQs, allowing us to explore specific mechanisms as potentiation targets. Aim 2 will enable us to understand how the coordination of DNA damage response changes due to carbon source availability after FQ treatment. We will deploy powerful genetic approaches and RNA sequencing in E. coli to interrogate the timing of gene expression and DNA repair. Completion of this aims will provide great insight into the importance of various DNA repair mechanisms in response to FQ treatment. Overall, the completion of these aims will reveal metabolic, biosynthetic, and expressional changes that underlie FQ persistence in E. coli. This information could illuminate potential genes that can be targeted to potentiate the activity of FQs and reduce resistance development in persister progenies. Additionally, these findings could lead to interesting implications for clinical infections, with a potential connection between host metabolism and antibiotic efficacy. This research could highlight the potential of adding glucose, as well as other metabolites, as supplemented compounds during antibiotic administration to improve the treatment of infections.