氧气在大肠杆菌稳定期持留菌形成中的作用机制研究

Persisters are a fraction of bacteria that can survive high dose of antibiotics due to epigenetic changes but not genetic mutations. Because the persisters can survive the antibiotic treatments and grow again when the antibiotics are removed, they are considered an important cause of prolonged treatment and relapse for various persistent bacterial infections. Persistence also could promote the generation of genetic resistance. Although significant progress has been made in our understanding of mechanisms of persister formation, the most significant observation of persister formation when bacteria enter stationary phase cannot yet be explained by our current understanding of persister mechanisms. In our recent studies, we made the very interesting observation that, after excluding other possible factors such as exhaustion of nutrients and toxic substance buildup, the cell density in stationary phase culture greatly affects the persistence phenomenon. RNAseq analysis showed that many genes related to hydrogen peroxide production were highly expressed when the stationary phase E.coli were diluted in its own supernatant. We found that intracellular hydrogen peroxide was significantly elevated at higher dissolved oxygen level, indicating that oxygen and hydrogen peroxide may play an important role in persister formation. Further investigation of the mechanisms of how oxygen and peroxide control the persister formation will pave the way to develop more effective drugs targeting persisters for more effective treatment of persistent bacterial infections.

持留菌是一种对抗生素不敏感的细菌，其本身并没有发生遗传物质变化，仅由于基因表达的变化形成一种能够在高浓度抗生素处理下依然存活的状态，在去掉抗生素之后能够重新恢复。由于持留菌的这种抗生素杀不死的特性，是造成一些感染性疾病治疗时间长、反复发作的重要原因，同时还会提高细菌产生耐药突变的风险。细菌进入稳定期是持留菌形成的最主要因素，但目前已经发现的持留菌形成机制并不能解释该现象。我们的前期研究发现，在排除了其它影响之后，稳定期的细菌密度降低之后会极大的增强抗生素的杀菌活性，RNAseq结果表明稀释之后细菌内部产生了大量的过氧化氢，而过氧化氢的产生与氧浓度升高相关，表明氧可能在稳定期细菌持留菌形成中起着关键的作用，本项目将对其作用机制进行深入研究，有望发现持留菌形成的重要机制，为开发能够持留菌药物，缩短感染性疾病的治疗时间奠定基础。

当对数期细菌生长到稳定期时，我们都知道持留菌的形成会达到高水平。然而，其中涉及的机制尚不清楚。我们假设抗生素耐受性可能与细胞密度的增加有关。我们将稳定期大肠杆菌培养物按照 1:100 稀释到其自身过滤的上清液中，以消除培养基中次生代谢物和信号模块的影响，并评估细菌密度对持留菌形成的影响。我们发现稀释的培养物对抗生素高度敏感，随后进行转录组学和代谢组学分析，我们发现氧化应激反应相关基因包括 ahpC、ahpF、katG、pspABCD、cysACHIJUW、grxA、trxC、ibpAB、hslUV 被广泛上调。此外，代谢组证实了谷胱甘肽的上调以及电子传递链受体 ADP/AMP/NAD/FAD 的下调。结果表明，由于细胞数量减少和氧气恒定，细菌在稀释后，暴露于溶解氧增加的培养基中。结果，代谢静止的细菌细胞被转化为活跃的代谢状态，从而导致对抗生素的高度敏感性。我们确定了 ΔahpC ΔahpF 和 ΔkatG 敲除菌株以及补充氧气的对照菌株的持留菌形成水平，并发现了氧气在杀死持留菌方面的协同作用。众所周知，氧气是能量代谢的关键因素，由于缺氧诱导因子介导哺乳动物细胞对氧气的感知和适应的机制被解开，其在传染病中的重要性越来越受到关注。尽管研究了几个世纪，但细菌如何感知氧气的具体机制及其在持久性形成中的作用仍然不清楚。我们的结果表明氧适应在大肠杆菌持留菌形成中的重要作用。