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

Abstract:

[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.