鱼类肠道粘附空间类型影响乳杆菌抗病力的分子机制研究

Intestinal adhesion is a selection criterion for fish dietary probiotics, however, the description on intestinal adhesion of probiotics in fish has been relatively primitive , and lacks the investigation on the spatial distribution in intestine (mucus vs mucosa), which limited the understanding and potential application of this probiotic trait. In this project, aided by metagenome, transcriptome, germ-free zebrafish & microbiota transfer techniques, and based on the three adhesion types classified in previous work, the applicant proposes to clarify the correlation between different intestinal adhesion types and disease resistance of Lactobacillus in fish, and select the responsive intestinal barriers, signaling pathways and sensitive markers, by using representative strains of the three adhesion types, i.e., the mucosa type strain L. rhamnosus GG (LGG), three strains Lactobacillus, the hybrid type strain L. brevis JCM1170 (JCM1170) and the mucus type strain L. rhamnosus JCM20300 (JCM20300). Moreover, systematic comparison will be carried out in wild type LGG and the isogenic pili-deficient LGG mutant strain by testing the differentiated pathways and barriers revealed above, which will provide insights in the molecular mechanism underlying the influence of Lactobacillus adhesion types on disease resistance. Overall, the results obtained in this study will guide the selection of fish dietary probiotics based on the gut adherence property, and lay the foundation for promoting antibiotic reduction and substitution in fish farming in China.

肠道粘附是鱼类饲用益生菌的优选指标之一，然而，目前对鱼类肠道粘附的认知还停留在抽象描述阶段，缺乏对肠道空间（如黏液vs黏膜）分布的考量，限制了对这一益生因素的深入理解与细分应用。本项目拟结合宏基因组、转录组、无菌鱼菌群转接等技术手段，以前期划分的乳杆菌斑马鱼肠道3大粘附空间类型为基础，依托各自代表株如黏膜粘附型鼠李糖乳杆菌GG（LGG）、混合型短乳杆菌JCM 1170及黏液型鼠李糖乳杆菌JCM 20300，在夯实乳杆菌鱼肠道粘附类型与抗病力之间的相关性基础上，筛选确定宿主应答肠道差异屏障层次及通路与敏感指标。进一步以LGG野生株及其粘附决定因子缺失突变株PB22为对象开展比较研究，结合敏感指标及差异通路与屏障层次论证分析，拟澄清鱼类肠道空间粘附类型影响乳杆菌抗病力的分子机制，以期为鱼类饲用益生菌基于肠道粘附的优选提供精准指导，为推进我国鱼类养殖减抗乃至替抗调控奠定基础。

本项目在原计划的基础上，以3 株模式乳杆菌为对象开展乳杆菌鱼类肠道粘附空间类型与抗病力之间的相关性及潜在分子机制探究。模式乳杆菌分别是包括黏膜型L. plantarum JCM1149（LP）、混合型L. brevis JCM1170（LB）和黏液型L. rhamnosus 20300（LR）。研究发现，饲喂混合黏附型乳杆菌LB浸浴保护力显著高于对照组与黏膜型乳杆菌LR（P<0.05)，也高于黏液型乳杆菌LP (P>0.05)。进一步发现饲喂3株均导致斑马鱼肠道菌群结构显著改变，但是混合型LB与黏膜型LP组改变幅度大于黏液型LR组。而且黏膜型菌株JCM1149诱导肠内炎症因子和Hsp70的表达高于其他菌株。进一步利用利用L. rhamnosus GG（黏膜型）及其缺乏SpaCBA菌毛的突变体PB22（混合型）表明，PB22抗病力强于LGG, 而且这是因为LGG造成斑马鱼肠道黏膜损伤。基于上述结果，进一步发现黏膜型与黏液型乳杆菌复配饲用策略与混合型乳杆菌具有相似的抗病力。项目澄清了鱼类肠道空间粘附类型影响乳杆菌抗病力的分子机制，并为基于肠道粘附的鱼类饲用益生菌复配策略提供指导。基于项目得到的传统益生菌在鱼类养殖的上的应用风险的提示，进一步推进了鱼类土著菌及相关益生元件的开发，加快推进我国鱼类养殖减抗替抗绿色发展。