PURIFICATION AND CHARACTERIZATION OF THIN, AGGREGATIVE FIMBRIAE FROM SALMONELLA-ENTERITIDIS
PURIFICATION AND CHARACTERIZATION OF THIN, AGGREGATIVE FIMBRIAE FROM SALMONELLA-ENTERITIDIS
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DOI：
10.1128/jb.173.15.4773-4781.1991
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发表时间：
1991-08-01
影响因子：
3.2
通讯作者：
KAY, WW
中科院分区：
文献类型：
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作者：
COLLINSON, SK;EMODY, L;KAY, WW
从人类肠道病原体肠炎沙门氏菌 27655 中分离和纯化出新的菌毛。这些菌毛很薄（直径为 3 至 4 nm），非常聚集，并且尽管尝试通过离心将它们与混合细胞分离，但仍保持细胞关联。 细菌毛不溶于 5 M NaOH 或沸腾的 0.5% 脱氧胆酸盐、8 M 尿素或含或不含 5% β-巯基乙醇的 1 至 2% 十二烷基硫酸钠 (SDS)。 因此，使用了一种非常规纯化程序，该程序基于通过酶消化和制备型 SDS-聚丙烯酰胺凝胶电泳 (PAGE) 去除超声处理的细胞提取物中的污染细胞大分子。 从凝胶孔中回收的不溶性菌毛需要在通过 SDS-PAGE 分析之前在甲酸中解聚。 酸解聚表明，菌毛由菌毛蛋白亚基组成，每个亚基的表观分子量为 17 kDa。 尽管它们的生化特征和氨基酸组成是一般菌毛的典型特征，但这些薄菌毛明显不同于其他先前表征的菌毛。 此外，它们的菌丝蛋白亚基具有独特的N端氨基酸序列。 全细胞上的天然菌毛用纯化菌毛产生的免疫血清进行特异性标记。 这种免疫血清还与蛋白质印迹中变性的 17-kDa 纤维蛋白发生反应。 多克隆免疫血清不与该菌株产生的其他两种天然菌毛类型或与蛋白质印迹（免疫印迹）上各自的菌毛蛋白发生交叉反应。 因此，这些菌毛代表了肠道病原体肠炎沙门氏菌产生的第三种菌毛类型。
Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.