460nm蓝光通过调控PVL- prophage基因抗创面MRSA感染的分子机制研究

As the multi-drug resistance grows, injured patients with MRSA infection are difficult to cure due to limited therapeutic options. Antimicrobial resistance is recognized as a global threat. Phototherapy appears to be potential alternative to antibiotic treatment. A growing number of studies have shown that light with a wavelength 460- 480nm, namely blue light (BL), has exhibited strong antibacterial effects. Although the antibacterial effect of BL has been investigated, but much less is known about the mechanism. Our previous study has shown that BL irradiation with 460 nm effectively eliminated biofilm MRSA and induced up-regulations of 164 genes (among those, 36 genes were phage-related) by RNA sequencing. Additionally, phage inhibitor GS-11P blocked the effect of anti-infection on MRSA after BL treatment. Therefore, we proposed that BL induces the SOS system and increases the protein level of RecA in MRSA. The prophage repressor CI can be inactivated by the increased enzyme RecA, leading to resumption of the lytic cycle of phages in MRSA. The lytic cycle results in the destruction of the infected MRSA by phage replications and BL kills MRSA. The aim of this study is to determine whether PVL-encoded prophages is involving in the modulation of BL anti-MRSA infection on skin wound in vitro and in vivo by inducing the prophages being lytic cycle (from lysogenic cycle). We, by completing the experiments, will elucidate the molecular mechanism of blue light anti-MRSA infection.

创面感染是烧／创领域亟待解决的难题之一，随着MRSA等耐药菌的出现抗感染治疗困境加剧。采用蓝光杀菌是具有潜力的治疗MRSA感染的措施，但其杀菌的机制仍有待阐明。我们前期发现460nm蓝光可杀灭细菌生物膜状态下MRSA, 并明显上调MRSA中噬菌体相关基因，噬菌体抑制剂可显著抑制蓝光杀菌效率。鉴于前噬菌体具有溶原和溶菌性周期的特征，我们推测蓝光可启动MRSA中SOS应答、活化RecA蛋白酶，使前噬菌体中CI阻遏蛋白失活，导致噬菌体终止溶原态、进入溶菌性周期，从而裂解杀灭MRSA，最终发挥抗感染作用。本研究拟通过分子生物学、遗传学、生物化学等手段，从体外至体内研究来验证以上假设。以期揭示蓝光杀菌的分子机制，为治疗创面MRSA感染提供理论基础和临床思路。

噬菌体是如何参与460 nm蓝光杀灭MRSA的机制尚不清楚，本课题首先通过明确了460 nm蓝光对MRSA的能量依赖的抑菌作用，确定半数致死量；在动物感染模型中，明确蓝光不影响正常皮肤细胞的活性，促进感染的皮肤伤口愈合；进一步验证了460 nm蓝光照射MRSA后，recA 表达下降，STAM2有效的和recA相互结合，从而达到蓝光抑菌效果；构建MRSA252- recA-gt11菌株，证实蓝光照射后MRSA252-recA-gt11菌株对蓝光的杀菌抑制作用有所缓解，验证recA在蓝光抑菌作用中起到重要作用。通过完成以上实验，揭示460nm蓝光对抗创伤后MRSA感染的作用机理，丰富蓝光杀菌的理论基础，进而为临床治疗创面MRSA感染提供新的思路和潜在靶点，具有重要的理论意义和临床价值。