原花青素抑制单增李斯特菌表面蛋白锚定的分子机制

Listeria monocytogenes, an gram positive bacterium responsible for several severe diseases in animals, has become a multifaceted model to investigate of bacterial infection and anti-infection. L. monocytogenes surface proteins, such as InlA, InlJ and LapB, play an critical role in L. monocytogenes internalization. Sortase A（SrtA）is a membrane enzyme responsible for the anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria. Therefore, new anti-infection strategies by targeting SrtA activity has attracted much attention in the word. Some natural compounds have been identified as effective inhibitors of SrtA. Among the tested compounds, procyanidins effectively inhibited SrtA activity at low concentrations. Molecular docking showed that procyanidins could hind the binding of SrtA to substrates by binding to the active site of SrtA. Based on previous results, this study is intended to further determine the protective effect of procyanidins against L. monocytogenes virulence in vitro and in vivo via cell-bacteria co-culture system and animal model of infection. Moreover, the molecular mechanism of procyanidins against SrtA activity would be characterized by molecular dynamics simulations, site directed mutation, fluorescence quenching method and microthermal swimming test. Together, this study would provide new leading compounds and novel strategy for the development of drug for antibacterial infection by targeting SrtA.

单增李斯特菌（LM）是兽医临床上重要的病原菌，也是开展细菌感染与抗感染研究的重要模式菌。有文献报道InlA、InlJ及LapB等多种蛋白在LM内化过程中发挥重要作用，而分选酶A（SrtA）是介导这些表面蛋白锚定到细菌细胞壁的关键催化酶。因此，通过抑制SrtA活性开展抗感染新思路的探索已经成为国内外关注的热点。申请人前期以单增李斯特菌SrtA为模型在天然化合物中进行筛选，发现原花青素及叶黄素等在较低浓度下可显著抑制SrtA的催化活性，其中原花青素的效果最好。分子对接研究发现原花青素结合于SrtA活性位点，阻碍酶与底物的结合。在此基础上，本项目拟通过分子动力学模拟、结合自由能计算、定点残基突变、荧光淬灭和微热量泳动在分子水平上阐明原花青素抑制SrtA活性的机制，并进一步在细胞水平和动物感染模型中验证原花青素对LM致病力的抑制作用，以期为以SrtA为靶标的抗细菌感染药物研发提供有效的先导化合物。

单增李斯特菌（LM）是兽医临床上重要的病原菌，也是开展细菌感染与抗感染研究的重要模式菌，其感染可引起动物的菌血症等多种致死性感染，给家畜养殖业造成重大经济损失。LM的致病因子较多，有研究表明SrtA和LLO等在LM感染及致病过程中发挥重要作用，是抗LM感染药物研发的重要潜在靶标。本项目以上述毒力因子为靶标，通过溶血试验、寡聚化试验、酶活性抑制试验及生物被膜抑制试验等，发现原花青素、染料木苷和穗花杉双黄酮等天然化合物在无抑菌活性浓度范围内可以有效抑制上述毒力因子的功能，通过细菌与细胞共培养感染体系中添加毒力因子活性抑制剂，显著降低细菌对宿主细胞的黏附与入侵，减缓细菌对宿主细胞的毒性作用；在小鼠感染模型中，经过活性抑制剂治疗后，可有效降低LM的致病力。本项目通过荧光猝灭、分子动力学模拟、结合自由能计算及定点残基突变等试验阐明了活性抑制剂与靶蛋白相互作用机制，发现抑制剂主要结合于靶蛋白的活性位点，阻碍靶蛋白与底物结合进而降低其生物学活性。综上，本项目为LM感染药物研发提供有效的先导化合物。