肠道分节丝状菌（SFB）鞭毛蛋白FliC3影响宿主特异性黏附机制研究

Host specific adhesion is a precondition for immunoregulation of the intestine by segmented filamentous bacteria (SFB). However, the mechanism for SFB specific adhesion is not clear. Our previous research showed that expression of SFB flagellin FliC3 could be detected in mouse intestine. Additionally, mouse SFB-FliC3 tended to bind to adhesion-related proteins from the mouse ileum more stable than rat SFB-FliC3. We also found that SFB-fliC3-egfp-Lactococcus lactis can adhere to the mice ileal mucosa by the host specific way. Therefore, we hypothesize that SFB-FliC3 is involved in the adhesion of SFB to the surface of the intestinal epithelial cells, and this adhesion has a host specificity. This project will determine host specificity for the adhesion of SFB-FliC3 using comparing cell models, and animal models including specific-pathogen-free and germ-free mouse. Additionally, we will use iTRAQ method to identify host proteins and signaling pathways that respond to the expression of SFB-FliC3. These protein-protein interactions will be validated using in vitro immunoprecipitation methods. This project will lay the foundation for further research on the specific adhesion and immunoregulation mechanisms of SFB.

宿主特异性黏附是肠道分节丝状菌SFB发挥免疫调控功能的前提条件。然而，SFB特异性黏附的分子机理并不清楚。本团队前期研究表明小鼠肠道内SFB表达鞭毛蛋白FliC3；小鼠SFB-FliC3比大鼠SFB-FliC3更稳定地结合小鼠回肠黏膜组织中黏附信号通路相关的蛋白；融合表达SFB-fliC3的乳酸乳球菌可以宿主特异性黏附在小鼠回肠末端黏膜上皮细胞。因此，我们推断SFB-FliC3参与SFB在肠道上皮细胞表面特异性黏附。本项目将分别采用细胞模型、SPF和无菌动物模型进一步验证SFB-FliC3的宿主特异性黏附功能；并通过iTRAQ定量蛋白组的方法筛选SFB-FliC3刺激引起的差异表达蛋白，找到应答SFB-FliC3的宿主效应蛋白与信号通路；最后，采用免疫共沉淀等方法对相互作用蛋白进行验证。本项目的实施将为深入研究SFB宿主特异性黏附的发生机理，以及进一步研究SFB免疫调控机制奠定基础。

肠道分节丝状菌（Segmented Filamentous Bacteria, SFB）通过与宿主回肠末端的肠上皮细胞特异性黏附而发挥重要的免疫调控功能。但对SFB的宿主特异性黏附机制还并不清楚。本团队前期研究发现：（1）SFB表达鞭毛蛋白FliC3, 且该蛋白序列具有物种特异性；（2）融合表达小鼠SFB-fliC3质粒的乳酸乳球菌黏附于小鼠回肠末端。我们推测：SFB FliC3参与SFB在宿主肠道上皮细胞的黏附，且具有宿主特异性。研究方法：（1）因未有成熟的SFB遗传操作系统而以乳酸乳球菌MG316S为工具菌，构建在MG316S菌中过表达的SFB flic3质粒，观察不同来源即大鼠（r）与小鼠（m）SFB flic3+MG316S菌与体外肠上皮细胞/或伪无菌小鼠肠上皮细胞的粘附；（2）分别通过体外表达纯化小鼠、大鼠来源SFB的 FliC3蛋白刺激实验小鼠，检测肠道固有层淋巴CD4+增殖为Th17细胞；（3）通过免疫荧光标记小鼠回肠末端组织FliC3蛋白抗体与内吞小体标志物共定位情况。结果：（1）（m）SFB flic3+MG316S 与（r）SFB flic3+MG316S菌株与宿主肠上皮细胞均有更强的粘附作用，但二者之间没有差异性；（2）不同来源的FliC3对肠道固有层淋巴CD4+增殖为Th17细胞有明显的促进作用，但二者之间没有差异性；（3）SFB flic3+定位在回肠末端上皮细胞顶端，但与早期内吞小体marker蛋白EEA1暂未观察到共定位。结论：SFB FLiC3可以促进菌体与宿主细胞的粘附作用， 以及促进肠道固有层淋巴CD4+的Th17细胞增殖，但SFB FLiC3的这些调控功能没有宿主特异性。SFB鞭毛蛋白FLiC3实现上述功能的基础可能是FLiC3可以作为抗原被宿主小肠上皮细胞提呈。后续将深入研究SFBFLiC3在宿主细胞的信号传递途径，包括进一步确定SFB- FLiC3是否通过内吞作用进入宿主细胞，以及SFB- FLiC3是否通过Ecadrin依赖途径的内吞作用进入。