ISOLATION AND CHARACTERIZATION OF 2 CAMPYLOBACTER GLYCINE-EXTRACTED PROTEINS THAT BIND TO HELA-CELL MEMBRANES
ISOLATION AND CHARACTERIZATION OF 2 CAMPYLOBACTER GLYCINE-EXTRACTED PROTEINS THAT BIND TO HELA-CELL MEMBRANES
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DOI：
10.1128/iai.61.8.3440-3448.1993
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发表时间：
1993-08-01
影响因子：
3.1
通讯作者：
FAUCHERE, JL
中科院分区：
文献类型：
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作者：
KERVELLA, M;PAGES, JM;FAUCHERE, JL
来自肠致病性弯曲杆菌的两种免疫原性蛋白27 （CBF1）和29 (CBF2) kDa似乎与哺乳动物细胞结合。通过酸萃取、制备凝胶电泳和电洗脱等方法，分别从病原菌和粘附菌空肠弯曲杆菌中纯化出这两种蛋白。制备了针对这些蛋白的兔多克隆抗血清。免疫学研究表明，CBF1对应PEB1， CBF2对应Pei等人（Z. Pei， R. T. Ellison， M. Blaser， J. Biol）描述的PEB4。化学。226:16363-16369,1991)。对具有抗CBF1、抗CBF2和抗PEB1的空肠C.粘附菌株进行免疫金标记，结果表明CBF1 （PEB1）表面暴露，而CBF2 （PEB4）表面不暴露。对14株菌株的全细胞提取物进行SDS -聚丙烯酰胺凝胶电泳（7 M尿素）和抗血清CBF1和CBF2免疫印迹分析表明，贴壁菌株和非贴壁菌株的CBF蛋白存在差异。使用纯化蛋白对细胞膜粘附的微测定表明，CBF1比CBF2具有更强的粘附性。CBF1抗血清可显著降低空肠梭菌对HeLa细胞的粘附，而CBF2抗血清是一种较差的抑制剂。纯化的CBF1竞争性地抑制了整个细菌对HELa细胞的粘附，而纯化的CBF2并不比牛血清白蛋白更好。在Tween 20或SDS的存在下，CBF2的依从性明显降低，而CBF1的依从性仅被SDS降低。我们得出结论：(i) CBF1 （PEB1）表面暴露，可能是空肠梭菌粘附的关键蛋白；（ii） CBF2 （PEB4）可能与CBF1络合，并在一定实验条件下与CBF1被动共粘附。贴壁菌株和非贴壁菌株含有这两种蛋白的不同同型，这可能是空肠梭菌粘附的有用标记。
Two immunogenic proteins of 27 (CBF1) and 29 (CBF2) kDa from enteropathogenic Campylobacter species appear to bind to mammalian cells. We purified these two proteins from a pathogenic and adherent Campylobacter jejuni strain to homogeneity by using acid extraction, preparative gel electrophoresis, and electroelution. Polyclonal rabbit antisera to these proteins were prepared. Immunologic studies indicate that CBF1 corresponds to the PEB1 and CBF2 corresponds to the PEB4 described by Pei et al. (Z. Pei, R. T. Ellison, and M. Blaser, J. Biol. Chem. 226:16363-16369, 1991). Immunogold labeling of a C. jejuni adherent strain with anti-CBF1, anti-CBF2, and anti-PEB1 suggested that CBF1 (PEB1) is surface exposed while CBF2 (PEB4) is not. Analysis of whole-cell extracts from 14 strains by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with 7 M urea and immunoblotting with antisera to CBF1 and CBF2 suggests that CBF proteins from adherent and nonadherent strains are different. Use of purified proteins in a microassay of adherence to cellular membranes indicated that CBF1 was much more adherent than CBF2. Adherence of C. jejuni to viable HeLa cells was markedly reduced with the antiserum to CBF1, whereas the CBF2 antiserum was a poor inhibitor. Purified CBF1 competitively inhibited adherence of whole bacteria to HELa cells, whereas purified CBF2 was no better a competitor than bovine serum albumin. Adherence of CBF2 was markedly reduced in the presence of Tween 20 or SDS, whereas adherence of CBF1 was reduced only by SDS. We conclude that (i) CBF1 (PEB1) is surface exposed and may be the key protein for C. jejuni adhesion and (ii) CBF2 (PEB4) may be complexed with CBF1 and may passively coadhere with CBF1 under certain experimental conditions. Adherent and nonadherent strains contain different isotypes of these two proteins which could be useful markers of C. jejuni adhesion.