水飞蓟宾抑制嗜水气单胞菌气溶素活性的分子机制

Aeromonas hydrophila (A. hydrophila) is an important bacterial pathogen of aquatic animals and human which threatened the healthy development of freshwater aquaculture and human health. The emergence of antibiotic resistant A. hydrophila strains limited the treatment of infections caused by A. hydrophila. Following infection, the secretion of aerolysin could lysis the membranes of red blood cells to provide nutrients for the growth of the bacteria. Moreover, aerolysin is necessary to induce cytokine secretion and apoptosis during infections. Aerolysin, as the key virulence factor, plays an indispensable role in the pathogenicity of A. hydrophila. Therefore, aerolysin is a promising potential targets for the development of anti-virulence drugs against A. hydrophila infection. In our previous study, we screened that silybin, a natural compound without anti-A. hydrophila activity, could reduce the pathogenicity of A. hydrophila via inhibiting the activity of aerolysin. Furthermore, our preliminary analysis was performed to analyze the potent binding site. Based on these findings, this study is intended to analyze the binding sites and the mechanism for silybin against the activity of aerolysin using molecular docking, molecular dynamics simulations. Moreover, fluorescence quenching, in vitro oligomer formation and analytical ultracentrifugation will performed to confirm the results. The therapeutic effects of silybin against A. hydrophila infection will be measured both in vitro and in vivo. Taken together, this study is expected to develop new fishery drugs targeting on aerolysin for healthy aquaculture in the future.

嗜水气单胞菌是一种重要的人-兽-鱼共患病原菌，耐药性菌株的出现严重制约了水产养殖嗜水气单胞菌感染的治疗。嗜水气单胞菌感染后通过分泌气溶素破坏宿主红细胞为细菌生长提供营养物质；此外气溶素可以引起上皮细胞分泌细胞因子甚至导致细胞凋亡。气溶素在嗜水气单胞菌致病过程中发挥着不可或缺的作用，因此气溶素是研究抗嗜水气单胞菌感染药物最具潜力靶标之一。本课题组通过建立相关的药物筛选平台，发现水飞蓟宾可以特异性的抑制嗜水气单胞菌气溶素的活性而降低其致病性，并初步分析了水飞蓟宾与气溶素结合的潜在氨基酸位点。本课题拟在前期研究基础上，应用分子对接、分子动力学模拟等试验技术分析水飞蓟宾抑制气溶素活性的结合位点和机制；通过荧光淬灭、体外寡聚体形成和分析超速离心试验确证分析结果；最后通过细胞保护性试验和治疗试验在体内外验证水飞蓟宾对嗜水气单胞菌感染的治疗作用。为以气溶素为靶标的新渔药研究和健康养殖奠定基础。

嗜水气单胞菌是淡水养殖鱼类的主要病原菌，耐药性菌株的出现制约了水产养殖嗜水气单胞菌感染的治疗，严重威胁了水产养殖业的健康发展。嗜水气单胞菌的致病性与其自身携带的毒力因子密切相关，研究发现气溶素在其感染构成中发挥了不可或缺的作用，是研究抗嗜水气单胞菌感染药物的潜在靶标。本项目发现桑色素、水飞蓟宾和木犀草素等天然化合物能直接抑制气溶素的活性，通过分子对接、动力学模拟、结合位点定点突变和荧光淬灭试验阐明了活性化合物抑制气溶素活性的分子机制并确证了主要结合位点，发现活性化合物可阻碍气溶素形成具有孔道的七聚体而降低靶蛋白的生物学活性。此外，本项目以嗜水气单胞菌气溶素为靶标，通过溶血试验、蛋白免疫印迹试验、荧光定量PCR试验和动物试验等发现厚朴酚、麝香草酚等天然化合物在无抗菌活性的浓度下可通过降低气溶素表达抑制嗜水气单胞菌的致病力。综上所述，本项目为水产养殖嗜水气单胞菌感染的新药研发提供了新策略和先导化合物。