Elucidating repair processes central to fluoroquinolone persistence in growth-inhibited populations

阐明对生长抑制人群中氟喹诺酮持久性至关重要的修复过程

• 批准号: 10409188
• 负责人: Allison Herzfeld
• 金额: $ 4.35万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10409188
• 关键词: Adjuvant; Antibiotics; Bacteria; Bacterial Infections; Biological Assay; Cells; Chemicals; Chromosomes; Chronic; Complement; DNA; DNA Damage; DNA Markers; DNA Repair; DNA Replication Timing; DNA biosynthesis; Data; Development; Dose; Drug Metabolic Detoxication; Drug Tolerance; Dyes; Enzymes; Escherichia coli; Excision; Exhibits; Fluorescence; Fluorescence Microscopy; Fluorescence-Activated Cell Sorting; Fluoroquinolones; Genetic; Goals; Gram-Negative Bacteria; Growth; Individual; Infection; Kinetics; Lead; Levaquin; Mediator of activation protein; Methods; Microbial Biofilms; Microscopy; Minimum Inhibitory Concentration measurement; Modeling; Modern Medicine; Moxifloxacin; Nucleic Acids; Nucleotides; Nutrient; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Population; Process; Pseudomonas aeruginosa; Recovery; Regulation; Relapse; Replication Origin; Reporter; Resolution; Rifampin; SOS Response; Savings; Site; Stains; Starvation; System; Temperature; Testing; Time; Treatment Failure; Work; antibiotic tolerance; bactericide; base; burden of illness; experience; experimental study; homologous recombination; inducible gene expression; mutant; novel; prevent; prophylactic; recombinational repair; repaired; soft drink; temperature sensitive mutant; time use; treatment duration

Project Summary Many antibiotics rapidly kill growing populations of bacteria but struggle to kill non-growing populations. Even for drugs that can kill the majority of growth-inhibited bacteria, such as fluoroquinolones (FQs), the presence of persisters can lead to treatment failure. While current paradigms suggest that persisters survive due to limited antibiotic-induced damage, for FQs this is not the case. In non-growing populations, FQ persisters experience the same amount of antibiotic-induced DNA damage as their genetically identical kin and require the homologous recombination repair machinery during the post-antibiotic recovery period in order to survive. Currently, the mechanism underlying why persisters can survive FQ-induced damage while their clonal kin cannot remains ill-defined. We hypothesize that, i) chromosome number, and ii) the relative timing of DNA synthesis and repair during the post-antibiotic period, are phenotypic variables that govern the likelihood a bacterium will be an FQ persister. Since our first hypothesis is based on the importance of homologous recombination to FQ persistence in growth-inhibited populations, we will use fluorescence-activated cell sorting (FACS) to sort live wild-type and mutant populations of Escherichia coli based on chromosome number as determined by staining with cell-permeant nucleic acid dyes, and will subject the isolated populations to tolerance assays and quantitative PCR for chromosome number verification. To complement these assays, we will use time-lapse microscopy of an FQ-treated E. coli strain that harbors an origin of replication reporter in order to visualize the chromosome content of persisters and nonpersisters during the post-FQ recovery period. Our second hypothesis is based on a recent study from our group that showed that starvation following FQ treatment increased persister levels in non-growing populations in a RecA- and time-dependent manner. To test whether the timing of DNA replication vs. DNA repair during recovery impacts FQ persistence, we will conduct time-lapse fluorescence microscopy of single cells harboring reporters for DNA repair or DNA replication both in the presence and absence of nutrients. We will then conduct bulk culture experiments by employing temperature sensitive mutants and inducible systems of the DNA replication and DNA repair machinery. We will first investigate levofloxacin, a representative FQ, and stationary-phase E. coli cultures, because non-growing infections are the most difficult to eradicate, before establishing the generality of any findings by using other FQs (e.g., moxifloxacin) and bacterial species (e.g., Pseudomonas aeruginosa). Data from these experiments will assess whether chromosome number and the relative timing of DNA synthesis vs. DNA repair during recovery from FQ treatment are phenotypic variables important for FQ persistence. Increased understanding of persister survival tactics will open the door for the development of anti-persister strategies, which would reduce the burden of chronic and relapsing infections.

项目摘要 许多抗生素可以迅速杀死不断增长的细菌种群，但很难杀死不增长的种群。 即使是可以杀死大多数生长抑制细菌的药物，如氟喹诺酮类（FQs）， 持续存在可导致治疗失败。虽然目前的模式表明，坚持者生存 由于有限的地震引起的损坏，对于FQs，情况并非如此。在非生长群体中，FQ 坚持者经历了与他们的基因相同的亲属相同数量的过敏性诱导的DNA损伤， 在抗生素后恢复期需要同源重组修复机制， 生存目前，为什么坚持者可以生存的机制，而他们的克隆 亲属关系不能模糊不清。我们假设，i）染色体数目，和ii）DNA的相对时间 在抗生素后时期的合成和修复，是控制可能性的表型变量， 细菌将是一个FQ坚持者。由于我们的第一个假设是基于同源的重要性， 为了在生长抑制的群体中观察到FQ重组的持续性，我们将使用荧光激活细胞分选 流式细胞仪（FACS）根据染色体数目对大肠杆菌的活野生型和突变体群体进行分选， 通过用细胞渗透性核酸染料染色来确定，并且将使分离的群体经受 用于染色体数目验证的耐受性测定和定量PCR。为了补充这些分析，我们 将使用延时显微镜观察一个经过处理的E.携带复制起点报告基因的大肠杆菌菌株 以便在FQ后恢复期观察持留菌和非持留菌的染色体含量。 我们的第二个假设是基于我们小组最近的一项研究，该研究表明， 治疗以RecA和时间依赖性方式增加了非生长群体中的持续水平。到 测试恢复期间DNA复制与DNA修复的时间是否会影响FQ持久性，我们将 对携带DNA修复或DNA修复报告基因的单细胞进行延时荧光显微镜检查 在有营养和无营养的情况下复制。然后，我们将进行批量培养实验， 使用温度敏感突变体和DNA复制和DNA修复的诱导系统， 机械.我们将首先研究左氧氟沙星（一种代表性的FQ）和静止相E。大肠杆菌培养物， 因为非生长性感染是最难根除的，在建立任何普遍性之前， 通过使用其他FQs（例如，氟沙星）和细菌种类（例如，铜绿假单胞菌）。数据 从这些实验将评估是否染色体数目和DNA合成的相对时间与 从FQ治疗恢复期间的DNA修复是FQ持续性的重要表型变量。 对持续生存战术的理解的增加将为反持续生存的发展打开大门 这些战略将减少慢性和复发性感染的负担。