| Shiga toxin-producing Escherichia coli (STEC) has caused many outbreaks and sporadic cases in the world, and has become serious public health and safety issues. It, especially enterohemorrhagic E. coli (EHEC) O157:H7, can cause human diarrhea and severe hemorrhagic colitis (HC), even the hemolytic uremic syndrome (HUS). Currently, cases infected by non-0157STEC are more frequent.In recent years, breakthroughs in research of the virulence gene structure and function in EHEC O157:H7have been achieved. And the whole genomes of four E. coli O157:H7strains have been sequenced. Of the four isolates, EDL933contains the main virulence genes:the stx gene encoded by A,-bacteriophagh, eaeA located in LEE pathogenicity island responsible for A/E phenotype and the hemolysin gene (hlyA) encoded by plasmid pO157. Shiga toxin is one of the most important virulence factor to human HC and HUS. Shiga toxin is divided into two subtypes: Stx1and Stx2, encoded by different phages which inserted into different locations of the genome in STEC. Most of EHEC O157:H7strains harbour both of the two subtypes of Shiga toxin, such as EDL933and Sakai, but Stxl is absent in the STEC O157:H7strain85m-1-2collected in our laboratory.PCR and sequence analyses showed that the strain85m-1-2harboured VT1bacteriophage but lost stxl, and the phage integrated into the chromosome and flanked by gene yehV. Sequences of the bacteriophage-yehV flanking genes represented high homology (99%) with the corresponding genes of EDL933deposited in the GenBank. Except for the deletion of stxl, both the upstream and downstream genes of stxl existed.In stx1+/stx2+EHEC O157:H7isolates, studies on the effect of eaeA, stx2and hlyA on pathogenicity were extensive, while rare attentions were involved in stx-/stx2+STEC O157:H7isolates. In this study, the mutants85mâ–³eaeA,85mâ–³stx2,85mâ–³hlyA and85mâ–³eaeAâ–³stx2â–³hlyA were constructed by allelic exchange. The plasmid pGEX-6P-1-stx2was electroporated into85mâ–³stx2and85mâ–³eaeAâ–³stx2â–³hlyA to generate the revertant Re85mâ–³stx2and Re85mâ–³eaeAâ–³stx2â–³hlyA. The results of RT-PCR demonstrated that the deletion of the gene eaeA, stx2and hlyA only disrupted the transcription of the target gene, while the upstream gene and the downstream gene of the target gene were transcripted normally.The biological characteristics of the wide-type strain, mutants and revertants were investigated. The growth curves of85mâ–³eaeA,85mâ–³stx2,85mâ–³hlyA and85mâ–³eaeAâ–³stx2â–³hlyA were similar to that of their parental strain85m-1-2in LB broth. The result of Hep-2cell adherence test showed that the adhesion ability of85mâ–³eaeA and85mâ–³eaeAâ–³stx2â–³hlyA was reduced compared with that of their wild-type strain. The mutants85mâ–³stx2and85mâ–³eaeAâ–³stx2â–³hlyA lost their cytotoxicity to Vero cell, and the cytotoxicity were restored in Re85mâ–³stx2and Re85mâ–³eaeAâ–³stx2â–³hlyA. The result of hemolytic test showed that the wild-type strain can form hemolytic ring on5%sheep bood agar surrounding the colonies, while the mutant85mâ–³hlyA and85mâ–³eaeAâ–³stx2â–³hlyA lose the ability.The lethal test in BALB/c mice showed that the mortality caused by strain85m-1-2(80%) isolate was slightly lower than that of EDL933(90%), and the death for the former was postponed. The mortality caused by85mâ–³eaeA was the same as that of85m-1-2, and the death for the former was also postponed, and85mâ–³hlyA (70%) caused the lowest mortality among them. Meanwhile,85mâ–³stx2and85mâ–³eaeAâ–³stx2â–³hlyA caused no deaths, while the mortality caused by two revertants was100%. After challenging, the mice inoculated with85m-1-2and EDL933excreted challenging bacteria from feces until two weeks later post-challenge. The excretion period of85mâ–³stx2last for11d,85mâ–³hlyA last for12d,85mâ–³eaeA and85mâ–³eaeAâ–³stx2â–³hlyA only last for7d using them as inocula respectively. |
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