鸡白痢沙门氏菌Peg菌毛受体的筛选与功能研究

The fimbriae binding to the surface receptor of host cells is the key step in the adhesion of Salmonella to the host, and is also a crucial factor for the host specificity and tissue tropism of the pathogen. Our preliminary studies demonstrated that the Peg fimbrial gene in Salmonella Pullorum was highly conserved, and its expression product could enhance the adhesion and colonization of Salmonella Pullorum to the chicken intestine, but the in-depth mechanism remains unclear. We hypothesized the existence of adhesion receptor in chicken intestinal epithelial cells, and inhibition of the adhension receptor expression will have an important influence on the pathogenicity of the pathogen. To test the hypothesis, we propose to use ligand blot assay labeled by viable organism to analyze the differentiated bands between the wild-type Salmonella and Δpeg mutant (constructed in the preliminary studies) while their binding to the chicken intestinal epithelial membrane proteins, followed by confirmation of the receptor protein and gene sequences by mass spectrometry and database retrieval. Furthermore, we will use RNA interference to inhibit the expression of the receptor, analyze its impact on the internalization of bacterial adhesion, cell damage，cell apoptosis and inflammatory response. This project will reveal the role of Peg fimbriae receptor in Salmonella Pullorum infection, provide a theoretical basis for further understanding of the interaction between the pathogen and host cells, and provide a new strategy for prevention and control of pullorum disease.

菌毛与宿主细胞表面受体的结合是沙门氏菌在宿主体内黏附定植的关键步骤，也是决定病原宿主特异性与组织亲嗜性的重要因素之一。申请者前期研究表明，Peg菌毛编码基因在鸡白痢沙门氏菌中高度保守，且Peg菌毛具有促进病原在鸡肠道黏附定植的作用，但其确切机制尚不清楚。申请者推测“Peg菌毛在鸡肠上皮细胞存在黏附受体，抑制受体表达对病原感染过程将产生重要影响”。为证实以上假说，本研究在已构建Peg菌毛缺失株的基础上，采用活菌标记的配基印迹技术，筛选野生株、缺失株与鸡肠上皮细胞膜蛋白的差异结合条带，通过质谱分析与数据库检索确定受体蛋白及其基因序列，并验证受体结合活性；利用RNA干扰技术抑制受体表达，分析其对细菌黏附内化、细胞损伤与凋亡、细胞炎性反应等产生的影响。本项目将揭示Peg菌毛受体在鸡白痢沙门氏菌感染过程中的作用，为深入理解病原与宿主细胞的相互作用提供理论依据，并为寻找有效的鸡白痢防治方法提供新思路。

菌毛与宿主细胞表面受体的结合是沙门氏菌在宿主体内黏附定植的关键步骤。沙门氏菌可以编码多种菌毛，其中少数菌毛（如Peg菌毛）仅存在于特定血清型中。为了筛选鸡白痢沙门氏菌Peg菌毛受体，并研究其生物学功能，本研究首先通过荧光定量PCR确定Peg菌毛基因在TSB肉汤中表达水平最高；以鸡白痢沙门氏菌标准株CVCC539为亲本株，通过Red同源重组系统构建了peg菌毛操纵子缺失株；将鸡白痢沙门氏菌野生株、缺失株分别与鸡源肠上皮细胞膜蛋白、猪源肠上皮细胞系IPEC-J2膜蛋白进行Ligand Blot反应后，都出现了差异结合条带，表明Peg菌毛受体没有宿主特异性；将CVCC539 Peg菌毛黏附素基因pegD克隆至大肠杆菌进行表达纯化，与LMH细胞膜蛋白进行Ligand Blot反应，将反应条带进行质谱分析；根据质谱结果，选取其中可信度，丰度均较高，且与电泳图谱中条带大小接近的疑似受体蛋白ATPа进行表达纯化；将PegD菌毛黏附素蛋白与ATPа蛋白分别进行ELISA与Ligand Blot实验，结果表明二者具有较强的结合活性；根据ATPа蛋白序列，设计并筛选了有效RNA干扰片段，通过抑制LMH细胞ATPа蛋白表达，发现对细菌黏附内化，细胞损伤与细胞凋亡有显著影响。本研究揭示了Peg菌毛受体在沙门氏菌感染过程中的作用，为深入理解病原与宿主细胞的相互作用提供理论依据。