婴儿源屎肠球菌表面蛋白E5拮抗单增李斯特菌黏附定植的作用研究

The interaction between symbiotic microorganisms and pathogenic bacteria is a hotspot but difficulty point for studying in molecular microecology. As the first batch of infant intestinal colonization, the probiotic or pathogenic properties of Enterococcus is one of the critical factors for the balance of host flora system and development of immune system for infants. There are quite fewer studies on the competition for adhesion site of probiotic Enterococcus to intestinal epithelial cells. In our previous work, we isolated a strain of Enterococcus faecium WEFA23 from baby infants and proved it to have none virulence genes and phenotypes, it can colonize intestinal tract and reduce serum cholesterol. Remarkably, its surface protein E5 can inhibit the adhesion of Listeria monocytogenes, however, its mechanism is not clarified. This project intends to adopt bioinformatics analysis and gene knockout technology to obtain a mutant strain of E5 from the wild strain of Enterococcus faecium WEFA23, the wild and mutant strains as well as E5 surface protein were compared for the effect on the adhesion of Listeria monocytogenes, with the aim to disclose the mechanism of E5 against the pathogen or its toxins, also to ascertain the potential immunity enhancement in mice. The expected results will be meaningful for providing experimental basis for the effective use of probiotic bacteria in infection prevention and control of food-borne pathogens.

肠道共生微生物与病原菌的相互作用是分子微生态学的研究热点与难点。作为首批婴幼儿肠道定植菌，肠球菌的益生/致病两重性对机体的微生态环境构建、免疫系统发育成熟起着特别关键作用。益生肠球菌与病原菌竞争、黏附肠道上皮细胞，行使益生功能，国内外研究颇少。前期工作中，分离获得一株婴儿源屎肠球菌WEFA23，其无毒力基因和表型，能定植肠道且能降低人体血清胆固醇，初步发现其表面蛋白E5能竞争抑制单核增生李斯特菌的菌体黏附，其作用机制亟待阐明。本项目拟采用生物信息学分析和基因敲除技术，获得屎肠球菌WEFA23的表面蛋白缺失突变株，在细胞水平上研究黏附蛋白定位、野生株和突变株拮抗单增李斯特菌的定植及毒素表达，对两者在小鼠体内对致病菌的拮抗和肠道免疫力进行探究。预期成果将发为有效利用益生肠球菌防控致病菌感染，提供实验基础。

肠球菌作为肠道共生微生物的代表，与食源性致病菌以及宿主肠道相互作用研究国内外鲜见，其表面蛋白参与拮抗单核增生李斯特黏附定植肠上皮细胞以及小鼠肠道，发挥生物学作用的分子机制尚未知，具有重要科学探索价值。.本文采用活体成像、荧光定量PCR、ELISA、免疫组化和激光共聚焦等多种技术手段，揭示了婴儿源屎肠球菌WEFA23表面蛋白E5拮抗单增李斯特菌黏附定植的分子机制。主要结果有：.1）采用生物信息学分析和基因敲除手段，以屎肠球菌WEFA23表面蛋白E5的重要组分鸟氨酸氨甲酰基转移酶（ornithine transcarbamylase，OTC）为研究对象，获得了屎肠球菌WEFA23的OTC基因敲除株；2）细胞水平研究发现，E5可显著降低单增李斯特菌的内化能力、炎症因子（IL-6和TNF-α）和毒力因子（inl A, plc A, plc B, act A和prf A）的转录水平（P < 0.05）；E5对增加跨膜电阻，提高紧密连接蛋白转录水平，以及减少FITC-Dextran含量，均具有显著性（P < 0.05）且呈量效关系；3）OTC基因敲除株与野生株的小鼠灌胃对比实验结果显示，E5可显著减少小鼠脏器（胃、盲肠和结肠）菌载量（P < 0.001）；改善小鼠肠道绒毛低矮、隐窝结构不明显（乃至消失）等症状，增加结肠杯状细胞，MUC2和ZO-1蛋白的含量；显著降低肝脏指数和肝脏中的单核巨噬细胞含量（P < 0.05）以及血清中IgA和IgG水平（P < 0.05）。.科学意义：屎肠球菌WEFA23的E5蛋白一方面通过减轻致病菌的毒力因子表达下降，修复并增强小鼠肠道屏障，减少了致病菌内化肠上皮细胞以及易位脏器的菌载量，另一方面通过降低机体炎症因子水平，改善炎性浸润，提高机体免疫力。从而起到恢复小鼠体重，减少小鼠死亡率的功效。以上结果为进一步探究人源/食品源的益生菌的功能组分拮抗食源性致病菌的侵染机制，促进机体健康的科学应用提供了思路和借鉴。