金黄色葡萄球菌sdh操纵子影响持留菌形成的机制研究

Bacterial persisters is one of the major reasons for the failure of clinical antibiotic therapy . Although persisters is first discovered 70 years ago in Staphylococcus aureus, no systematic research has been carried out on this field. Through building and screening transposon insertion mutants library of S. aureus, the author found 129 mutations in 18 genes which had a decreased persister level. By gene knockout and complementation, it was verified that ΔsdhAB in vitro resulted in 10-400 times decreased levels of persisters under various pressures. A hypothesis was proposed that sdh operon affect S. aureus persister formation by bacterial electron transport chain rather than energy metabolism. By gene knockout and complementation experiments, persistence assay under both aerobic and anaerobic conditions, persisters experiments in mice models, bacterial virulence experiments in mice model and micro-organisms phenotype microarray, the proposal first aims to comprehensive test and verify role and influence of sdh operon on persisters formation phenotype and other related phenotype. Secondly, the effects and molecular mechanisms of how the electron transport chain genes including sdh influence the persisters formation will be further clarify through four experiments: electron transport chain related gene double knockout, RNA-seq, HITS and uses of electron transport chain inhibitors. The study is expected to uncover an important mechanism for persisters formation of Staphylococcus aureus, to explicitly provide new drug targets and to support a theoretical basis for the clinical treatment of chronic infections caused by persisters.

持留菌是临床抗生素治疗失败的重要原因之一。尽管70年前最先在金葡菌中发现了持留菌，但到目前为止在金葡菌中未开展过系统性的研究。申请人前期通过构建和筛选金葡菌转座子插入突变库，找到了129个克隆包含18个持留菌形成缺陷的候选基因，通过基因敲除和回补验证了sdhA/B缺失在体外导致持留菌水平下降10-400倍。推测sdh操纵子影响持留菌的形成可能通过电子传递链而非能量代谢。本研究拟开展基因敲除和回补、有氧和无氧持留菌实验、小鼠持留菌实验、小鼠毒力实验、微生物表型芯片等技术，全面检测与验证sdh操纵子对持留菌形成以及其它表型的影响。然后再通过电子传递链相关基因双敲除、RNA-seq、HITS技术和使用电子传递链抑制剂等实验进一步明确包括sdh在内的电子传递链基因对细菌持留菌形成的影响和分子机制。该研究有望明确金葡菌持留菌形成的一个重要机制，提供新的药物靶标以及对持留菌的临床治疗提供理论依据。

本研究首次通过插入突变库筛选研究找到了15个可能与金黄色葡萄球菌持留形成相关的基因，其中sdhA/B、ndh、ctaB、hemA等均属于细菌的电子传递链相关基因，在其他抗生素、高温、酸性等多种压力条件下生存显著降低，提示电子传递链基因可能参与细菌持留生存。该两部分内容刚发表在2015年Frontiers in Microbiology杂志上.进一步通过构建ΔsdhCAB和ΔsdhAB的敲除和回补菌株，开展细菌持留研究，观察到sdhCAB缺失后对细菌的持留表型的缺陷，说明sdhCAB操纵子对细菌的持留有着重要作用。这一部分内容刚发表在2018年微生物与感染杂志上。.本研究还发现呼吸链相关基因hemA和hemB是持留菌形成中的重要基因，通过构建敲除株后，观察到了细菌生长的缺陷以及对各种压力条件下的生存缺陷，说明这两个基因对细菌的持留具有重大影响。进一步通过RNA-seq研究发现，敲除hemA和hemB的敲除株的基因表达差异主要位于碳水化合物代谢通路，氨基酸代谢通路，辅因子和维生素代谢通路，膜转运体以及能量代谢通路。在进一步通过构建双敲除株和回补突变验证了与hemA相关的部分基因对持留菌的影响。该部分内容已完成论文撰写，正在投稿中。.本研究有助于揭开革兰氏阳性细菌持留菌形成的重要机制之一，为持留菌的清除提供了新的药物靶标，对持留菌导致的慢性感染的临床治疗提供理论依据。.研究期间发表标记基金好的SCI论文6篇，中文核心期刊1篇，另有一篇英文论文在投稿，培养研究生一名。