Schmidt H, Beutin L, Karch H
Institut für Hygiene und Mikrobiologie, Universität Wüzburg, Germany.
Infect Immun. 1995 Mar;63(3):1055-61. doi: 10.1128/iai.63.3.1055-1061.1995.
In this study, we determined the nucleotide sequence of the 5.4-kb SalI restriction fragment of the recombinant plasmid pEO40-1, cloned from the large plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933. This revealed two open reading frames which shared approximately 60% homology to the hlyC and hlyA genes of the E. coli alpha-hemolysin (alpha-hly) operon. We termed these genes EHEC-hlyA and EHEC-hlyC to distinguish them from the alpha-hly genes. Preliminary sequence analysis indicated that another open reading frame homolog to the hlyB gene is located close to the 3' end of EHEC-hlyA. The predicted molecular masses of the EHEC-hlyA and EHEC-hlyC gene products were 107 and 19.9 kDa, respectively. The EHEC hemolysin protein (EHEC-Hly) was not secreted into the culture supernatant by the strain EDL 933. However, hemolytic activity was found in the broth culture supernatant after transforming EDL 933 with the recombinant plasmid pRSC6 carrying the hlyB and hlyD genes from the E. coli alpha-hemolysin operon. The EHEC hemolysin was precipitated and used as an antigen for immunoblot analysis. This demonstrated that 19 of 20 reconvalescent-phase serum samples from patients with hemolytic uremic syndrome reacted specifically with the antigen; conversely, only 1 of 20 control serum samples demonstrated reactivity. To investigate the prevalence of EHEC hemolysin genes in diarrheagenic E. coli, a PCR was developed to specifically detect EHEC-hlyA. All Shiga-like toxin-producing O157 strains and 12 of 25 Shiga-like toxin-producing non-O157 strains were PCR positive; strains of other categories of diarrheagenic E. coli were PCR negative. All PCR-positive strains hybridized with the CVD 419 probe. We found the CVD 419 probe to be identical to the 3.4-kb HindIII fragment of plasmid pEO40 carrying most of the EHEC-hlyA gene and a part of the putative EHEC-hlyB gene. In this study, the newly discovered EHEC hemolysin was shown to be responsible for the enterohemolytic phenotype and demonstrated to be related but not identical to alpha-hemolysin. The EHEC hemolysin appears to have clinical importance because it occurs in all O157 strains tested and is reactive to sera of patients with hemolytic uremic syndrome.
在本研究中，我们测定了从肠出血性大肠杆菌（EHEC）O157:H7菌株EDL 933的大质粒中克隆的重组质粒pEO40 - 1的5.4kb SalI限制性片段的核苷酸序列。这揭示了两个开放阅读框，它们与大肠杆菌α - 溶血素（α - hly）操纵子的hlyC和hlyA基因具有约60%的同源性。我们将这些基因命名为EHEC - hlyA和EHEC - hlyC，以将它们与α - hly基因区分开来。初步序列分析表明，另一个与hlyB基因同源的开放阅读框位于EHEC - hlyA的3'端附近。EHEC - hlyA和EHEC - hlyC基因产物的预测分子量分别为107 kDa和19.9 kDa。EDL 933菌株未将EHEC溶血素蛋白（EHEC - Hly）分泌到培养上清液中。然而，在用携带大肠杆菌α - 溶血素操纵子的hlyB和hlyD基因的重组质粒pRSC6转化EDL 933后，在肉汤培养上清液中发现了溶血活性。EHEC溶血素被沉淀下来并用作免疫印迹分析的抗原。这表明，20份溶血尿毒综合征患者恢复期血清样本中有19份与该抗原发生特异性反应；相反，20份对照血清样本中只有1份显示出反应性。为了研究EHEC溶血素基因在致泻性大肠杆菌中的流行情况，开发了一种PCR方法来特异性检测EHEC - hlyA。所有产志贺样毒素的O157菌株和25株产志贺样毒素的非O157菌株中的12株PCR检测呈阳性；其他类别的致泻性大肠杆菌菌株PCR检测呈阴性。所有PCR阳性菌株均与CVD 419探针杂交。我们发现CVD 419探针与携带大部分EHEC - hlyA基因和部分推定的EHEC - hlyB基因的质粒pEO40的3.4kb HindIII片段相同。在本研究中，新发现的EHEC溶血素被证明是肠溶血表型的原因，并证明与α - 溶血素相关但不相同。EHEC溶血素似乎具有临床重要性，因为它存在于所有测试的O157菌株中，并且与溶血尿毒综合征患者的血清有反应。
