以femA为靶点探讨青蒿琥酯增强β-内酰胺类抗生素抗MRSA机制研究

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant bacteria leading to high mortality，it is important to find new antibiotics or antibiotic sensitizers for the treatment of MRSA. FemA is essential for the expression of high-level methicillin resistance in MRSA, drugs leading to femA inactivation seem to be a sensitizer to β-lactams. Our preliminary research found that artesunate could improve the sensibility of MRSA to β-lactams, and artesunate could inhibit the expression of femA in MRSA, and the structure and function changes of MRSA caused by artesunate are similar with the changes caused by femA inactivation. Therefore, we hypothesized that femA was the key target of mechanism of artesunate act on MRSA. In this study, we are aimed to improve the hypothesis., we would make femA-deletion mutants and femA+ backcross mutants, and compare the artesunate antibacterial sensitization effect, crosslink of cell wall peptidoglycan, glycine content of cell wall, autolysis rate and cell wall permeability in wild type, femA-deletion mutant and femA+ backcross mutant. Then,.we would pinpoint upstream regulatory proteins under the action of femA gene inhibition induced by artesunate, the direction effect of artesunate on femA gene sequence, the interaction between artesunate and femA protein, in order to get the target of artesunate inhibiting femA. The results of this study could provide theory basis for discovery of new effective sensitizers to antibiotics.

耐甲氧西林金葡菌(MRSA)具有多重耐药性，致死率高，寻找新的抗菌药物或抗菌增敏剂有重要意义。femA是MRSA关键耐药基因之一，抑制femA药物可增强β-内酰胺类抗生素抗MRSA活性。青蒿琥酯能增强β-内酰胺类抗MRSA活性，并可抑制MRSA femA的基因表达和蛋白水平，同时其导致的细菌结构功能改变与femA失活细菌的改变一致。我们推测影响femA可能是青蒿琥酯抗菌增敏效应主要机制。本项目拟以MRSA为研究对象，构建并比较MRSA野生菌、femA敲除菌和femA补回菌上青蒿琥酯的抗菌增敏效果、胞壁肽聚糖链长度、胞壁甘氨酸含量、细菌自溶速率及胞壁抗渗透能力变化，确证femA为青蒿琥酯抗菌增敏作用靶点；并通过观察青蒿琥酯是否对femA基因有直接作用，或影响主要启动区段调控蛋白，又或直接与femA蛋白作用，从而明确青蒿琥酯影响femA作用机制。本课题的实施将为更多有效抗菌增敏剂的开发奠定基础。

MRSA导致的感染是临床常见耐药菌感染，其耐药机制复杂。本研究按计划进行过程中出现了技术难题，意外发现青蒿琥酯可以抑制MRSA生物膜形成，因此微调本研究内容为青蒿琥酯对MRSA抗菌增敏机制的另一个分支：青蒿琥酯对MRSA生物膜形成的影响和可能的机制。MRSA的生物膜形成是其多重耐药的重要原因之一。抗菌药物在治疗由MRSA生物膜引起的细菌感染时较为困难，而针对这些感染的有效策略很少。我们的结果表明青蒿琥酯可以剂量依赖性方式显着抑制MRSA的生物膜形成。青蒿琥酯还可以抑制细菌黏附力，hla，RNAIII和agrA的表达以及α-溶血素的产生。青蒿琥酯对黏附的影响基因（clfA，clfB，fnbA，fnbB）具有菌株特异性，但不影响ica基因的表达。结果表明青蒿琥酯可能通过agr系统抑制α-溶血素合成，从而抑制生物膜形成。同时，我们进一步发现青蒿琥酯对MRSA诱导的小鼠肺炎模型具有保护作用，其联合苯唑西林组相对于单纯的苯唑西林组有明显的更优保护作用，并明显降低小鼠体内炎症水平。