Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis
Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis
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DOI：
10.1128/jb.184.12.3360-3367.2002
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发表时间：
2002-06-01
影响因子：
3.2
通讯作者：
Korhonen, TK
中科院分区：
文献类型：
--
作者：
Hynönen, U;Westerlund-Wikström, B;Korhonen, TK
从细菌表面去除SlpA蛋白质大大降低了短乳杆菌ATCC 8287对人肠细胞系Caco-2和肠407、内皮细胞系EA-hy 926和膀胱细胞系T24以及固定化纤连蛋白的粘附。对于SlpA表面蛋白的功能分析，将slpA基因的不同区域表达为大肠杆菌fliC（H7）基因可变区中的内部框内融合物。由此产生的嵌合鞭毛携带来自成熟S层蛋白（大小为435个氨基酸）的长达275个氨基酸的插入片段。嵌合鞭毛上的SlpA片段的表达通过免疫电子显微镜和Western印迹使用抗SlpA抗体进行评估，并通过间接免疫荧光评估它们与人细胞的结合。嵌合鞭毛窝藏插入物，代表的N-末端部分的S-层蛋白结合的上皮细胞系，而C-末端部分的S-层蛋白没有赋予鞭毛上的结合。能够检测到结合的最短S-层肽的大小为81个氨基酸残基，代表未加工S-层蛋白中的残基96至176。细菌和嵌合鞭毛未显示与红细胞的可检测结合，而表达SlpA的ATCC 8287细胞以及嵌合SlpA 96-245/FliC鞭毛与固定化纤连蛋白结合。融合到FliC的N-末端SlpA肽96-176或96-200在Western印迹或免疫电子显微镜中不被针对S-层蛋白产生的多克隆血清识别;然而，抗血清在免疫荧光中与ATCC 8287细胞反应。相反，针对SlpA的His-标记的肽96-245产生的抗血清结合到具有N-末端SlpA插入物的杂合鞭毛，但不与ATCC 8287细胞反应。结果确定了L. brevis ATCC 8287作为对人上皮细胞和纤连蛋白具有亲和力的粘附素，并将受体结合区定位在分子N-末端部分的81个氨基酸的片段内，其在天然S-层中似乎是抗体不可接近的。
Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.