Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli
Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli
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DOI：
10.1006/abbi.2001.2355
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发表时间：
2001-06-01
影响因子：
3.9
通讯作者：
Ganguly, NK
中科院分区：
文献类型：
--
作者：
Grover, V;Ghosh, S;Ganguly, NK
从肠聚集性大肠杆菌菌株中鉴定出菌毛粘附素。在FPLC系统中，通过在亲和基质和凝胶过滤柱上的连续层析将粘附素纯化至740倍。经等电聚焦电泳（pI 7.25）证实，纯化蛋白具有均一性。经Superose 12 HR 10/30柱凝胶过滤层析证实，天然粘附素为高分子量聚集蛋白。然而，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，发现粘附素的分子量为18 kDa，这通过在Superose 6 HR 10/30柱上存在6 M盐酸胍的凝胶过滤色谱进一步证实。N端15个氨基酸的序列与已报道的菌毛粘附素没有同源性。纯化的粘附素在Ca ~（2+）（5 mM）存在下与人红细胞有粘附作用，其最适温度为25 ℃，最适pH为6.5。抑制研究清楚地表明粘附素的结合位点可以识别半乳糖作为特异性糖。4-甲基伞形酮基-α-D-吡喃半乳糖苷与粘附素结合后荧光猝灭，用棉子糖竞争取代滴定法观察到荧光猝灭的最大逆转。结合常数和结合自由能分别为3.98 × 10（5）M-1和-31.97 kJ/mol。在半乳糖的存在下，细菌对HEp-2单层的粘附被抑制，并且这进一步得到了细菌对用β-D-半乳糖苷酶预处理的HEp-2细胞的粘附显著减少的支持，（C）2001 Academic Press。
A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain. The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system. The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25), The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride. The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins, The purified adhesin showed adhesion to human erythrocytes in the presence of Ca2+ (5 mM), The optimum temperature and pH for the hemadhesion activity was found to be 25 degreesC and 6,5, respectively. The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar. The fluorescence of 4-methylumbelliferyl-alpha -D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose, The adhesin was found to contain one binding site per monomer for its specific sugar residue. The association constant and the free energy of binding were obtained as 3.98 x 10(5) M-1 and -31.97 kJ/mol, respectively. The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta -D-galactosidase, (C) 2001 Academic Press.