Voss E, Paton A W, Manning P A, Paton J C
Molecular Microbiology Unit, Women's and Children's Hospital, North Adelaide, South Australia.
Infect Immun. 1998 Apr;66(4):1467-72. doi: 10.1128/IAI.66.4.1467-1472.1998.
Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli (STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H-, and reactivity to O111:H- whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H- cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin. The deduced amino acid sequence of the O111:H- intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H- STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H-. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H- STEC did react with serum from a patient infected with both O111:H- and O157:H- STEC.
采用蛋白质印迹分析评估了与一起由受产志贺毒素大肠杆菌（STEC）污染的发酵香肠引起的溶血尿毒综合征（HUS）暴发相关的恢复期患者血清的反应性。从HUS患者中分离出的主要STEC属于O111:H-血清型，并检测了其对经蛋白酶K处理或未处理的O111:H-全细胞裂解物的反应性。正如预期的那样，所有五个血清样本均表现出明显的抗脂多糖反应，但也有几条蛋白带具有免疫反应性，特别是一条表观大小为94 kDa的蛋白带。随后使用一份恢复期血清样本筛选O111:H-黏粒文库，发现900个黏粒克隆中有2个呈阳性，二者均含有相似的DNA插入片段。对其中一个克隆进行蛋白质印迹分析，鉴定出三条主要的免疫反应性蛋白带，大小约为94、70和50 kDa。所有五个恢复期血清样本均可检测到对这三种蛋白的免疫反应，但健康人血清则无此反应。免疫反应性94 kDa和50 kDa的蛋白由一个含有7 kb STEC DNA插入片段的黏粒缺失衍生物产生。该区域的序列分析表明，它是肠细胞损伤位点的一部分，包括编码紧密素的eaeA基因。O111:H-紧密素的推导氨基酸序列与O157:H7 STEC的紧密素的同一性为88.6%，差异最大的区域是羧基末端的200个残基，其同一性仅为75%。这种差异可能具有抗原学意义，因为仅感染O111:H- STEC的HUS患者的血清与来自肠致病性大肠杆菌O111菌株的紧密素以及其他几种eaeA阳性STEC分离株发生反应，但不与属于O157:H-血清型的eaeA阳性STEC发生反应。其他两名HUS患者的血清也未与后一种菌株的紧密素发生反应。然而，O157:H- STEC的紧密素确实与同时感染O111:H-和O157:H- STEC的患者的血清发生反应。
