致泻性大肠杆菌和福氏2a志贺氏菌染色体上毒力岛和基因组岛插入相关的tRNA位点的研究

Correlation of putative pathogenicity islands of diarrheogenic E.coli and Shigella flexneri 2a with abnormal tRNA induced by genomic insertion with pathogenicity islands

目的探讨毒力岛及外源DNA片段的插入与异常tRNA位点的相关性。方法采用PCR和杂交的方法,从异常tRNA位点寻找致泻性大肠杆菌和志贺氏菌的福氏2a菌基因组中可能的毒力岛或基因组岛。以Escherichia coli K12MG1655序列中的86个tRNA位点的45个区域设计引物,对与K12亲缘较近的几类菌株的相关tRNA及其临近序列的完整性进行了初筛,对发现异常者进一步从其内部补充引物扩增证实,并联合杂交方法进行验证,以已知的E.coli O157∶H7EDL933和S.flexneri2a301的序列来对比PCR结果。结果菌株EDL933,S.flexneri2a301,EPEC2348/69,ETEC10407,EIEC8401,E.coliO157∶H78823/64,EAEC O42,E.coli O25F171中异常tRNA位点的个数分别是14,18,15,14,15,11,21,19。通过序列比对EDL933中8个tRNA位点有O岛插入,S.flexneri2a301中有6个插入了毒力岛,其余的位点均有外源片段插入或原有片段缺失。结论毒力岛及外源DNA片段的插入与异常tRNA位点密切相关。

To explore the correlation of putative pathogenicity islands （PAIs） of diarrheogenic E. coli and Shigella flexneri 2a wih abnormal tRNA induced by genomic insertion with pathogenicity islands, the primers used in PCR were designed from 45 regions of 86 tRNA loci in the MG1655 sequence of E. coli K12 strain, and isolates of E. coli and Sh. flexmeri 2a were screened for the putative tRNA loci that were integrated by PAIs or genomic islands. The efficiency of this PCR screening method was supported by the genomic sequence data of E. coli O157, H7 strain EDL933 and Sh. flexneri 2a strain 301 published. In 14 integrated tRNA loci of E. coli strain EDL933 as suggested by PCR screening , 8 loci had so-called O-island inserted, whereas, in 18 integrated tRNA loci suggested for Sh. flexneri 2a strain 301, 6 loci were integrated by putative pathogenicity islands. The rest loci had either exogenous insertion with small gene fragments or deletion of the original gene fragments. For other tested isolates of diarrheogenic E. coli, 15, 14, 15, 11, 21 and 19 putative intergated tRNA loci were observed in enteropathogenic Escherchia coli （EPEC） strain 2348/69, enterotoxigenic Escherchia coli （ETEC） strain10407, enteroinvasive Escherchia coli （EIEC） strain 8401, E. coli O157 ： H7 stain 8823/64, enteroadhesive Escherchia coli （EAEC） strain 042 and E. coli 025 F171 respectively. It is thus concluded the pathogenicity islands and the exogenous insertion of DNA fragments are closely related to abnormal tRNA loci.