兔肠致病性大肠杆菌lifA基因的免疫调节和黏附特性

兔肠致病性大肠杆菌(rEPEC)菌株RDEC-1的基因组中lifA基因与LEE(Locus for enterocyte effacement)致病岛相毗邻.本试验通过DNA序列分析、基因打靶技术、细胞因子检测以及动物试验,分析lifA基因完整核苷酸序列及其生物学功能.结果表明,RDEC-1的lifA基因的核苷酸序列与人肠致病性大肠杆菌的完全相同;ifA基因具有降低家兔外周血单核细胞IL-2表达的作用.与野生型菌株RDEC-1相比,被定点敲除lifA基因的RDEC-1突变株(RDEC-1△lifA)口服接种家兔后,排菌量明显降低.利用野生型RDEC-1和RDEC-1△lifA基因缺失菌株同时口服接种家兔,从粪便中分离细菌,结果显示野生型RDEC-1是优势菌,而RDEC-1△lifA基因缺失菌数量极少.RDEC-1△lifA基因缺失菌株和野生型RDEC-1都能引起特征性家兔肠道上皮的黏附与细胞脱落病变(A/Elesion).表明rEPEC的lifA基因在免疫调节和细菌的肠道定居中起重要作用,这为研究lifA基因的生物学功能提供了直接证据.

Lymphostatin (lifA) of rabbit enteropathogenic Escherichia coli possesses both immunomodulation and adhesion properties

The rabbit enteropathogenic E. coli (rEPEC) strain RDEC-1 possesses a lifA homologue adjacent to the LEE pathogenicity island. To study the entire nucleotide sequence and biological function of lifA,the DNA sequence and biological function of RDEC-1 lifA were analysed with gene cloning,gene knock-out and in vivo virulence examination. The result showed that the entire coding sequence of the lifA of RDEC-1 shares nearly absolute homology with the lifA of human isolates. RDEC-1 lifA inhibited IL-2 expression in stimulated rabbit peripheral blood mononuclear cells. We further demonstrated significant reduction in fecal bacterial shedding by RDEC-1 derivative lifA mutant when compared with its parent strain. In a competitive study when rabbits were inoculated with a combination of the WT and the mutant, the WT was the predominant bacteria recovered from fecal samples, while fewer mutant bacteria were recovered. However,the lifA mutant is able to induce A/E type of lesions as efficient as the parent strain. The data provide direct evidence that lifA of rEPEC plays a role in immunomodulation and in in vivo colonization in the intestinal tract.