EHEC O157:H7 z3276基因缺失株构建及其生物学特性

Construction and characterization of z3276 gene knocked-out mutant of EHEC O157:H7

【目的】肠出血性大肠杆菌O157:H7是世界范围内重要的动物源性致病菌之一,可感染人。I型菌毛是多种致病性大肠杆菌(如肾盂肾炎型大肠杆菌等)可表达的一种黏附结构,与细菌吸附黏膜表面密切相关。然而,O157:H7 fim操纵子上几个核苷酸的缺失却导致其不能表达I型菌毛。BLAST比对结果表明O157:H7独有的开放阅读框z3276编码的氨基酸序列与其他大肠杆菌I型菌毛高度同源,这可能是对O157:H7不能表达I型菌毛的补偿机制,但确切功能尚不清楚。本文探究z3276基因的生物学功能。【方法】利用O157:H7 86-24参考菌株构建z3276基因缺失株(?z3276),并构建其互补株(C?z3276),进而比较亲本株、?z3276与C?z3276的生物学特性及对小鼠致病性差异。【结果】与亲本株相比,?z3276进入对数生长期的时间延后,在半固体琼脂平板上的迁移直径明显缩小,生物被膜形成能力显著减弱。?z3276对HEp-2细胞的黏附和侵袭能力并无明显变化,但对IPEC-J2细胞的侵袭能力明显减弱。在小鼠攻毒试验中,?z3276组排菌数量减少、排菌持续时间缩短。C?z3276各项特性均能回复到与亲本株一致的水平。【结论】z3276基因可能是O157:H7重要的毒力相关因子。

[Objective] Enterohaemorrhagic Escherichia coli（EHEC） O157：H7 is a worldwide important zoonotic pathogen that causes foodborne infections in humans. Type I fimbriae are important bacterial adhesion organelles present in various types of pathogenic E. coli（e.g. Uropathogenic E. coli） that facilitate bacterial colonization. However, EHEC O157：H7 cannot express type I fimbriae because of base deletion in the fim operon. BLAST analysis shows that the open reading frame z3276, a specific genetic marker of EHEC O157：H7, encodes a sequence with high amino acid identity to other E. coli type I fimbriae. It is possible that z3276-encoding protein is a compensatory mechanism for type I fimbriae, but its definitive function in EHEC O157：H7 remains unclear. We explore the biological function of z3276 gene. [Methods] We targeted the reference EHEC O157：H7 86-24 strain to construct a z3276 mutant（？z3276） and its complement strain（C？z3276）, and the biological characteristics and pathogenicity of ？z3276 in mice were compared with the parental strain. [Results] Motility and biofilm formation assays revealed a smaller twitching motility zone for ？z3276 on the agar surface and significantly decreased biofilm formation by ？z3276 compared with the parental strain. The adhesion and invasion ability of ？z3276 to HEp-2 cells showed no significant change, but the invasion ability of ？z3276 to IPEC-J2 cells was attenuated. During mouse colonization, we observed shortened and lower fecal shedding for ？z3276 compared with the parental strain. [Conclusion] Thus, z3276 appears to be a crucial virulence factor of EHEC O157：H7.