The Dynamics of DNA in Salmonella Persisters in Macrophages

摘要 细菌的顽固性导致抗生素治疗失败和许多顽固者复发 感染。持久菌是暂时不生长的细菌的一个亚群，能够在抗菌剂中存活下来 来自抗生素和免疫系统的攻击，最终恢复生长。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在 巨噬细胞，在那里它们能存活很长时间。研究表明，虽然沙门氏菌不生长，但 持久者保留在巨噬细胞中表达和注射效应蛋白的能力，从而导致干扰 宿主的免疫反应和支持持久生存。尽管如此，坚持不懈的人仍然容易受到 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久者也活跃地复制 染色体DNA，可以积累四个以上的染色体等价物的DNA。我发现 持久者在生长停止的情况下复制完整的染色体，这种染色体扩增是 与持续性再生的频率较高有关。我假设遇到的压力是 巨噬细胞进入触发一种特定的生长停滞状态，在这种状态下非典型染色体复制被启用 然后通过同源重组促进染色体DSB的修复。为了评估这一假设，我将 破译沙门氏菌DNA合成的机制和后果。在《目标1》中，我将 表征染色体扩增对同源重组的贡献，从而持久 生死存亡。我将使用基因转换试验来测量高DNA持久者的同源重组 内容(1.1)。然后，我将通过使用以下工具跟踪DSB修复来评估DSB修复如何影响周围再生 DNA损伤反应的荧光成像和转录报告(1.2)。在目标2中，我将决定 在生长停滞的情况下DNA合成的基础，包括启动DNA合成和 巨噬细胞内触发这种非典型DNA合成的条件。我将确定以下要求 通过微染色体复制分析在ORIC启动染色体复制(2.1)。我将评估 巨噬细胞通过评估基因上持久者的DNA积累来触发非典型DNA复制 改变的巨噬细胞(2.2)。总之，这项研究将进一步加深我们对细胞内持久者的理解 包括持久状态的形成、维持和再生长。对持久力的机械理解 生存最终将有助于开发针对持久者的方法，增强抗生素 药效，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.