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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要许多抗生素可以快速杀死不断增长的细菌群体，但很难杀死非增长的细菌群体。即使对于能够杀死大多数生长抑制细菌的药物，例如氟喹诺酮类药物（FQ），持续存在可能导致治疗失败。虽然当前的范式表明坚持者能够生存由于抗生素引起的损害有限，对于 FQ 来说情况并非如此。在非增长人口中，FQ 坚持者经历与他们基因相同的亲属相同数量的抗生素引起的 DNA 损伤在抗生素后恢复期间需要同源重组修复机制，以便存活。目前，持久性细胞能够在 FQ 引起的损伤中幸存下来，而其克隆细胞却能幸存下来的机制亲属关系不能一直模糊不清。我们假设，i) 染色体数量，ii) DNA 的相对时间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成为 FQ 持久者。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群体中实现 FQ 持久性重组，我们将使用荧光激活细胞分选 (FACS) 根据染色体数目对活的野生型和突变型大肠杆菌群体进行分类通过用可渗透细胞的核酸染料染色来确定，并对分离的群体进行用于染色体数目验证的耐受性测定和定量PCR。为了补充这些测定，我们将使用延时显微镜观察经过 FQ 处理的大肠杆菌菌株，该菌株具有复制起点报告基因为了可视化 FQ 后恢复期间持久性和非持久性的染色体含量。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 FQ 后的饥饿治疗以 RecA 和时间依赖性方式增加非增长人群中的持久性水平。到测试恢复过程中 DNA 复制与 DNA 修复的时间是否会影响 FQ 持久性，我们将对带有 DNA 修复报告基因或 DNA 的单细胞进行延时荧光显微镜检查在有营养物和无营养物的情况下都可以复制。然后我们将进行批量培养实验采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 FQ）和稳定期大肠杆菌培养物，因为在确定任何感染的普遍性之前，非生长性感染是最难根除的使用其他 FQ（例如莫西沙星）和细菌种类（例如铜绿假单胞菌）得出的结果。数据这些实验将评估染色体数量和 DNA 合成的相对时间是否与染色体数量和 DNA 合成的相对时间有关。 FQ 治疗恢复期间的 DNA 修复是对 FQ 持久性很重要的表型变量。对持久者生存策略的更多了解将为反持久者的发展打开大门战略，这将减轻慢性和复发感染的负担。