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The Biological Characteristics and Shiga-toxin1Expression Regulation of EHEC Variant

Author: DaiShiZuo
Tutor: LiZuo
School: Wuhan Polytechnic University
Course: Microbial and Biochemical Pharmacy
Keywords: Enterohemorrhagic Escherichia coli Shiga toxin (Stx) phage induction RT-real time PCR hiTAIL-PCR gene clone and sequencing protein Q prokaryotic expression
CLC: R378
Type: Master's thesis
Year: 2012
Downloads: 23
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Abstract


Enterohemorrhagic Escherichia coli(EHEC) is an important food-borne pathogen thatcan cause severe gastrointestinal and systemic diseases such as hemorrhagic colitis(HC),hemolytic uremic syndrome(HUS)and thrombotic thrombocytopenic purpur(TTP), andeven cause death. The pathogenesis of EHEC, especially serotype O157:H7, is highlysophisticated. There are many virulence factors that contribute to the pathogenicity ofEHEC strains, including stx, eae, hly and ehxA genes. The major disease determinant forEHEC is Shiga toxin (Stx) produced in the intestine. Two immunologically distinct typesof Stx, Stx1and Stx2, can be produced by EHEC.My laboratory has isolated a kind of rare EHEC variant, which carried stx1and stx2genes but could not produce Shiga-toxins. Currently, only a few laboratories in Japan gaveout similar research reports, but the regulation mechanism of Shiga-toxin expression in theEHEC variant still remained unknown.In the present study, we analyzed the biological characteristics of this kind of EHECvariant, and made a primary study in the Shiga-toxin1expression regulation of EHECvariant.The Shiga toxin titers of two EHEC variant strains, EC130and EC169, weremeasured by reversed passive latex agglutination (RPLA) assay. It proved that the twostrains did not produce Shiga-toxins. PCR assays showed that EC130and EC169carriedstxl, stx2, eae and hly genes. The results of PCR amplification of rfbO157and fliCH7genesand serological test showed that EC169could be determined as O157:H7serotype, whileEC130was non-O157serotype. The result of RAPD assay also demonstrated that thegenetic relationship was far between EC130and the other three O157:H7strains.Stx bacteriophages were induced by mitomycin C from EC169and EC130, purifiedby two-layer agar assay using the DH5as indicator. A large number of plaques could beobserved and it showed that the EHEC variants had released a lot of phage particlesinduced by mitomycin C.The stx1mRNA transcription of EC169was measured by relative quantitative RT-realtime PCR. We analyzed the RT-PCR result by CTcomparison method. The type strain EC1was used as a calibrator. The quantity of the RT-PCR products of stx1was normalizedagainst the16S rRNA gene product. EC169exhibited nearly undetectable level of stx1transcripts. It indicated that the stx1gene was not effectively transcribed in the EC169. The stx1gene and its upstream region of EC1and EC169were amplified by PCR.The PCR products were then cloned and sequenced. The upstream sequence of stx1inEC169was achieved by hiTAIL-PCR. The stx1sequences of EC1and EC169showed99%similarity to that of E. coli O157:H7standard strain Sakai. There was no any basesubstitution or base deletion in the stx1promoter. The failed production of stx1in EC169should not be caused by non-functional stx1promoter or SNP loci in the stx1gene. Thesequences of anti-terminator Q showed100%similarity between EC1and the type strainSakai, while EC169had6SNP sites in the q gene.The q genes of EC1and EC169were amplified by PCR, and cloned to ProkaryoticExpression System pET-28b(+) respectively. Then, the recombinant plasmid wastransformed into Escherichia coli BL21(DE3). The expression of protein Q was induced byIPTG. Then the whole-cell proteins of EC130, EC169and the two recombinant strainswere extracted and used for SDS-PAGE. The result showed that the expression of proteinQ was improved significantly after IPTG induction. Protein Q could be expressed in bothEC1and EC169.This study demonstrated that EC169and EC130could release phages after mitomycinC induction. And the phages released might transfer to E. coli strains again by lysogenicinfection, and might form new pathogens by expressing Shiga-toxins. The6SNP sites ofthe q gene of EC169might result in weak anti-termination activity of the Q protein,subsequently, lead to the low replication and transcription levels of the stx1gene. Thisresearch provided basic data for the gene regulation and pathogenicity of the EHECvariants, which were stx-positive but failed to produce Stx.

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