桑色素抑制产气荚膜梭菌NetB活性的分子机制

Clostridium perfringens is the main causative agent of chicken necrotic enteritis (CNE), which usually affected broilers during the withdrawal period, seriously threatening the healthy development of the poultry industry. Clostridium perfringens produces a variety of virulence factors and necrotic enteritis B-like toxin (NetB), a pore-forming toxin that lyses intestinal epithelial cells, is an essential virulence factor associated with avian necrotic enteritis caused by Clostridium perfringens. We have previously isolated various pathogenic Clostridium perfringens strains and identified the gene encoding NetB in these strains. Some natural compounds have been identified as effective inhibitors of NetB by hemolysis assay, in which morin has the highest inhibitory activity. Based on previous results, this study is intended to further determine the protective effect of morin against Clostridium perfringens virulence in vitro and in vivo via cell-bacteria co-culture system and chicken model of infection. Moreover, the molecular mechanism of morin against NetB activity would be characterized by molecular dynamics simulations, site directed mutation and MST. Together, this study would provide new leading compounds and novel strategy for the development of drug for chicken necrotic enteritis, especially those during the withdrawal period.

产气荚膜梭菌是鸡坏死性肠炎的主要致病菌，一般在肉鸡养殖抗生素休药期内易发，严重威胁养鸡业的健康发展。产气荚膜梭菌致病因子较多，有文献报道NetB可裂解肠上皮细胞并导致坏死性肠炎，是与鸡坏死性肠炎直接相关的重要毒力因子。本课题组前期研究中已在国内分离到多株致病性产气荚膜梭菌，并扩增到NetB编码基因，进一步实现了NetB的原核表达和纯化，根据NetB成孔活性过程中其空间构象变化特性，进行了抑制剂的筛选并获得了多种有效天然化合物，其中以桑色素抑制活性最强。在此基础上，本项目拟通过化学生物学相关实验阐明桑色素和NetB相互作用的分子机制；细胞感染实验和实验动物感染实验分别在体内外验证桑色素对产气荚膜梭菌感染的防治作用，以期为防治鸡坏死性肠炎，尤其是休药期内鸡坏死性肠炎的新药研发提供新的策略和先导化合物。

产气荚膜梭菌是鸡坏死性肠炎的主要致病菌，一般在肉鸡养殖抗生素休药期内易发，严重威胁养鸡业的健康发展。产气荚膜梭菌致病因子较多，有研究表明NetB、IV型菌毛系统、PFO和α毒素等在细菌感染过程中发挥着不可或缺的作用，是抗产气荚膜梭菌感染药物研发的重要潜在靶标。本项目以上述毒力因子或IV型菌毛系统为靶标，通过溶血试验、细菌滑行运动试验和生物被膜形成抑制试验，发现桑色素、鸢尾黄素和穗花山双黄酮等天然化合物在无抗菌活性浓度范围内可有效抑制上述毒力因子或IV型菌毛系统生物学功能，在细胞与细菌共培养感染系统中，加入活性抑制剂显著降低细菌对宿主细胞的粘附作用及其对宿主细胞介导的细胞毒性作用；在小鼠或肉鸡感染模型中，经活性抑制剂治疗后，可有效降低产气荚膜梭菌致病力。另外，本项目通过化学生物学试验、定点参加突变和荧光淬灭等试验阐明和确证了活性抑制剂与靶蛋白互作机制，发现抑制剂主要结合于靶蛋白活性中心位点，阻碍靶蛋白与其底物结合而降低靶蛋白生物学活性。综上，本项目为产气荚膜梭菌感染，尤其是休药期内鸡坏死性肠炎的新药研发提供新型综合策略和先导化合物。