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

项目摘要 许多抗生素迅速杀死了不断增长的细菌数量，但却很难杀死没有增长的细菌数量。 即使对于可以杀死大多数生长抑制细菌的药物，如氟喹诺酮类药物(FQS)， 持久者的存在可能导致治疗失败。虽然目前的范式表明，持久者会活下来 由于抗生素引起的损害有限，对于FQS来说情况并非如此。在不增长的人口中，FQ 持久者经历的抗生素引起的DNA损伤与他们的基因相同的亲属和 在抗生素后恢复期要求同源重组修复机制 活下去。目前，持久者能够在FQ诱导的损伤中幸存下来的机制，而他们的克隆 亲属不能一直定义不清。我们假设，i)染色体数目，ii)DNA的相对时序 抗生素后时期的合成和修复是控制可能性a的表型变量 细菌将成为FQ的持久者。因为我们的第一个假设是基于同源的重要性 重组到FQ持久性在生长抑制的人群中，我们将使用荧光激活的细胞分选 (FACS)根据染色体数目对野生型和突变型活菌群进行排序 通过细胞穿透核酸染料染色确定，并将使隔离的种群 耐受性分析和定量聚合酶链式反应验证染色体数目。为了补充这些化验结果，我们 将使用FQ处理的大肠杆菌菌株的延时显微镜，该菌株含有复制报告的起始点 以直观地显示FQ后恢复期内持续者和非持续者的染色体内容。 我们的第二个假设是基于我们团队最近的一项研究，该研究表明FQ之后的饥饿 治疗以一种依赖于RecA和时间的方式增加了非增长人群中的持久水平。至 测试恢复期间DNA复制和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报告的单细胞进行延时荧光显微镜检查 在营养物质存在和不存在的情况下进行复制。然后，我们将通过以下方式进行大规模养殖试验 利用DNA复制和修复的温度敏感突变体和诱导系统 机械设备。我们将首先研究具有代表性的FQ左氧氟沙星和固定相大肠杆菌培养物， 因为非增长感染是最难根除的，在建立任何 使用其他FQS(例如莫西沙星)和细菌种类(例如铜绿假单胞菌)的结果。数据 这些实验将评估染色体数量和DNA合成的相对时间与 FQ治疗后恢复过程中的DNA修复是FQ持久性的重要表型变量。 增加对持久生存策略的理解将为反持久生存策略的发展打开大门 战略，这将减轻慢性和复发性感染的负担。

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.