Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Environmental and Life Sciences, C. Norwida 31, 50-375 Wrocław, Poland.
Division of Biochemistry, Institute of Organic Chemistry, Biochemistry and Biotechnology, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
Microbiology (Reading). 2010 Jun;156(Pt 6):1738-1748. doi: 10.1099/mic.0.039206-0. Epub 2010 Mar 18.
The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.
肠沙门氏菌血清型肠炎和鼠伤寒（肠炎和鼠伤寒）的低亲和性和高亲和性 FimH 黏附素的结合特性使用包含代表 FimF 蛋白 N 端延伸的额外肽的嵌合蛋白进行了研究。这种修饰利用供体链交换机制封闭了 FimH 黏附素纤毛结构域中的疏水性凹槽。这种自我互补的黏附素（scFimH）不会形成聚集体，并且比天然 FimH 更稳定（抵抗蛋白水解切割）。通过对来自低亲和性肠炎和鼠伤寒变体的 fimH 基因进行定点突变，获得了 scFimH 蛋白的高亲和性变体，其中位置 61 为丙氨酸，位置 118 为丝氨酸，而位置 61 为甘氨酸，位置 118 为苯丙氨酸。使用表面等离子体共振（SPR）和携带高甘露糖碳水化合物链的糖蛋白（RNase B、辣根过氧化物酶和甘露糖-BSA）进行直接动力学分析表明，不仅在鼠伤寒中，而且在肠炎中都存在高亲和性和低亲和性等位基因变体。使用来自肠炎的低亲和性 FimH 蛋白的另外两个突变体（Gly61Ala 和 Phe118Ser），SPR 分析表明 Ser118 是肠炎 1 型菌毛高亲和性表型的主要决定因素。这些研究首次证明，在整个菌毛细菌中观察到的功能差异可以在纯化的黏附素水平上重现。它们强烈表明，1 型菌毛的黏附特性仅由 FimH 蛋白的结构差异决定，而不受黏附素所在的菌毛轴的影响。
