与HEp-2细胞粘附现象相关的大肠杆菌O157基因的发现

Genes of Escherichia coli O157 Associated with Adherence to HEp-2 Cells

将产志贺毒素大肠杆菌O157∶H7菌株97094培养于含有不同质量浓度的萘啶酮酸(nalidixicacid,NA)的麦康凯(MacConkey)琼脂平板上,获得对NA有抗性的菌株。用含有自杀性质粒pRT733的供体菌SM10λpir(携带转座子TnphoA)和抗萘啶酮酸的97094菌株做接合试验,使转座子TnphoA转入受体菌97094,经过约200个接合试验,有503个菌株在含有Km、NA、XP的LB琼脂平板上形成蓝色菌落,说明在这些菌株中形成了phoA融合基因,并能表达phoA基因。在这503个Kmr,NAr变异体中,有6株完全失去了对HEp-2细胞的局灶型粘附(localadherence,LA)现象。对构建的6株大肠杆菌O157TnphoA变异体,克隆TnphoA及其两侧的大肠杆菌O157DNA序列,用根据TnphoA融合接点处的phoA的DNA序列设计合成的寡核苷酸引物,对各克隆中TnphoA上游的大肠杆菌O157的DNA片段部分进行测序,测序结果做BLAST搜索,发现有4个变异体的TnphoA插入到已知的大肠杆菌O157紧密素基因eae中,有2个变异体的TnphoA分别插入到eae之外的核苷酸序列中,分别插入到一个功能未知的菌毛分子伴侣基因和一个功能未知的Z4182基因的上游。这项研究表明,除紧密素外,大肠杆菌O157尚有其他因子参与对真核细胞的粘附。

Shiga toxin-producing Escherichia coli O157strain 97094 was cultured in MaConkey agar medium containing nalidixic acid(NA),and NA resistant strains were selected.Conjugation test was performed with suicide plasmid-containing donor strain SM10λpir(with transposon TnphoA) and NA resistant E.coli O157 strain.In about 200 conjugation tests,A total 503 strains produced blue colonies in the LB medium containing Km,NA,and XP.This result showed that the phoA fusion gene was produced and the phoA gene was expressed in these mutants.The adherence of these 503 strains to HEp-2 cells was tested,and 6 of them lost the local adherence to the cells.Each TnphoA insert along with flanking E.coli O157 sequences was cloned.The cloned DNA portion of E.coli O157 was sequenced using the oligonucleotide primer that hybridizes to phoA sequences just downstream of the fusion joint.The sequences were searched for their homology using BLAST.TnphoA was found to have inserted to eae in four mutants,to ycbR gene in one mutant and between the start point and the-10 sequence of Z4182 gene in another mutant.YCBR are a putative fimbrial chaperone required for the biogenesis of the putative YCBQ fimbia,and the function of Z4182 gene is unknown.These data suggest that YCBR and the product of Z4182 gene are associated with adherence of E.coli to epithelial cells in vitro.