The Dynamics of DNA in Salmonella Persisters in Macrophages

摘要 细菌的持续存在导致抗生素治疗失败和许多复发性腹泻 感染.持续性细菌是一种暂时不生长的细菌亚群，能够在抗微生物药物作用下存活。 抗生素和免疫系统的攻击，并最终恢复增长。许多病原体包括， 肠道沙门氏菌，结核分枝杆菌和金黄色葡萄球菌，形成持久性内 巨噬细胞在那里存活很长时间。结果表明，尽管沙门氏菌不生长， 存留细胞保留了在巨噬细胞中表达和注射效应蛋白的能力，导致干扰巨噬细胞， 宿主免疫反应和支持持续生存。尽管如此，坚持者仍然容易受到 巨噬细胞以双链断裂（DSB）的形式诱导DNA损伤，并需要DSB修复 通过同源重组。引人注目的是，巨噬细胞内的沙门氏菌持留菌也积极复制 染色体DNA，并且可以积累超过四个染色体当量的DNA。我发现 坚持复制完整的染色体，尽管生长停滞，这种染色体扩增， 与更高频率的持续再生有关。我推测， 巨噬细胞进入触发生长停滞的特定状态，其中非典型染色体复制被启动， 则有利于通过同源重组修复染色体DSB。为了验证这个假设，我将 破译沙门氏菌persisters中DNA合成的机制和后果。在目标1中，我将 描述染色体扩增对同源重组的贡献，从而持续 生存我将使用基因转换分析来测量高DNA含量的持续者中的同源重组 内容（1.1）。然后，我将评估如何DSB修复影响持久再生跟踪DSB修复使用 DNA损伤反应的荧光成像和转录报告基因（1.2）。在目标2中，我将确定 尽管生长停滞，但DNA合成的基础包括DNA合成起始的要求， 引发这种非典型DNA合成的巨噬细胞内条件。我将确定以下要求： 通过微型染色体复制试验在oriC启动染色体复制（2.1）。我将评估 巨噬细胞通过评估遗传学上持续存在的DNA积累来触发非典型DNA复制， 改变巨噬细胞（2.2）。总之，这项研究将进一步加深我们对细胞内持久性的理解 包括形成、维持持久状态和再生长。对持久性的机理性理解 生存将最终有助于发展针对持久性，增强抗生素 有效性，并防止抗生素耐药性的发展。

Abstract Bacterial persistence contributes to antibiotic treatment failure and the relapse of many recalcitrant infections. Persisters are a subpopulation of transiently non-growing bacteria capable of surviving antimicrobial attacks from antibiotics and the immune system and eventually resuming growth. Many pathogens including, Salmonella enterica, Mycobacterium tuberculosis, and Staphylococcus aureus, form persisters within macrophages where they survive extended periods of time. It was shown that, although non-growing, Salmonella persisters retain the ability to express and inject effector proteins in macrophages leading to interference with the host immune response and supporting persister survival. Nonetheless, persisters remain vulnerable to macrophage-induced DNA damage in the form of double stranded breaks (DSBs) and require DSB repair through homologous recombination. Strikingly, intramacrophage Salmonella persisters also actively replicate chromosomal DNA and can accumulate more than four chromosome equivalents of DNA. I have found that persisters replicate complete chromosomes despite growth arrest and that this chromosome amplification is associated with a higher frequency of persister regrowth. I hypothesize that stresses encountered upon macrophage entry trigger a specific state of growth arrest where atypical chromosome replication is enabled and then favors repair of chromosomal DSBs by homologous recombination. To evaluate this hypothesis, I will decipher the mechanisms and consequences of DNA synthesis in Salmonella persisters. In Aim 1, I will characterize the contribution of chromosome amplification to homologous recombination and thus persister survival. I will use gene conversion assays to measure homologous recombination in persisters with high DNA content (1.1). I will then assess how DSB repair affects persister regrowth by tracking DSB repair using fluorescent imaging and transcriptional reporters of the DNA damage response (1.2). In Aim 2, I will determine the basis for DNA synthesis despite growth arrest including the requirements for initiation of DNA synthesis and intramacrophage conditions that trigger this atypical DNA synthesis. I will determine the requirements for initiation of chromosome replication at oriC through minichromosome replication assays (2.1). I will assess the macrophage triggers for atypical DNA replication by evaluating DNA accumulation of persisters in genetically- altered macrophages (2.2). Altogether, this research will further our understanding of intracellular persisters including formation, maintenance of the persistent state, and re-growth. Mechanistic understanding of persister survival will ultimately contribute to the development of approaches for targeting persisters, enhancing antibiotic efficacy, and preventing the development of antibiotic resistance.