O157:h7 Ehec Z3672 Gene Knockout And Toxicity Evaluation

The prevalence of pathogenic microorganisms is a major threat to human health. Elucidating the molecular and cellular mechanisms underlying bacteria-host interactions and understanding the origination and evolution of these mechanisms are crucial for understanding the relationship between humans and microbes, as well as for preventing and treating infectious diseases and improving human health. As a new subject, bioinformatics which is widely used in all fields of life science researches, is playing a significant role. Comparative genomics have emerged to study the sequences of pathogenic microorganisms that are related with pathogenesis with more complete genome sequences are known, so that the study of pathogenic microorganisms are more effectively. In the previous study, we discovered a new protein family (FlxA-like proteins) in bioinformatics methods. The proteins of this family are suspected to involve in the interactions between pathogens and their hosts, and may be important effectors (virulence proteins) of pathogenic bacteria with type III secretion system.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attachingand effacing (A/E) lesions. It causes serious illness known as diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombo- cytopenic purpura, and severe cases can lead to death, with the death rate of 5% to 10%. In 1982, Riley reported an outbreak of hemorrhagic colitis caused by the O157: H7. This was the first time that O157: H7 is recognized as a serious pathogen. And prevalence in Canada, Japan, Britain, Australia are reported from then. In 1999, O157 broke out in Jiangsu and Anhui Province and lasted for 7 months. More than 20,000 people were infected, and 177 patients died. Therefore, EHEC is known as one of the 12 pathogenic bacteria which may cause serious illness in China. O157: H7 which can cause significant morbidity and mortality worldwide, has become a global problem of public health. O157 which cultured easily, spread via many different ways, and cause many serious disease. Less than 100 CFU (colony forming units) can cause infections. Thus, O157 is classified as B class bioterrorism pathogens by Centers for Disease Control (CDC). In the last 30 years, much of the mechanism of O157: H7 are characterized but is still not clearly understood. It is important to improve our ability of preventing and treating the outbreak of disease caused by O157: H7.In the previous study, we discovered a new protein family (FlxA-like proteins) in bioinformatics methods. The O157: H7 Z3672 protein is a member of this family. Being a hypothetical protein, Z3672 is suspected to involve in the interactions between pathogens and their hosts and secret by type III secretion system. Therefore, O157: H7 is expected to be a new effector of typeⅢsecretion system. It may be helpful to clarify the molecular mechanisms underlying the host-microbe interactions with understanding of the function of EHEC O157: H7 Z3672 protein. Also, it will be useful for our further understanding of FlxA-like protein family in host-microbe interaction.To examine the function of Z3672, we constructed z3672 deletion mutant strain O157:H7Δz3672. Mutation in z3672 gene of O157:H7 was constructed by the PCR one-stepλred recombinant system. First of all, we amplified z3672 gene homologous sequences from O157:H7 genome, and then inserted them into pET-24a. We get a long-arm homologous sequence of z3672, which had 500bp homologous sequence in both ends. The homologous sequences were electroporated into O157:H7 recipient strains carrying the Red system expression plasmid pKOBEG, and mutants were selected on LB plates with kanamycin. The strain of z3672 deletion mutant was verified by PCR and sequencing. At last, the z3672 deletion mutant strain O157:H7Δz3672 was constructed. The Real-Time PCR assay was applied to determine whether z3672 is transcribed in O157: H7 wide type. We are sure of that z3672 is transcribed in O157: H7 wide type.On one hand, to characterize the Z3672 function, a z3672 mutant and the wild-type O157:H7 strains were assessed for two dimensional (2D) electrophoresis and LC/MS/MS to characterize the Z3672 function. In O157: H7 z3672 mutant, the expression of a outer membrane protein (OmpA) is increased. Then, Real-Time PCR assay was applied to determine the transcription level of OmpA in both z3672 mutant and the wild-type O157:H7 strains. It also demonstrated that the transcription of OmpA is increased in z3672 mutant. So, z3672 gene may affect in the expression of OmpA (outer membrane protein A). On the other hand, a z3672 mutant and the wild-type O157:H7 strains were assessed for A/E lesion formation in vitro. Infecting HeLa cells with the mutant and the wild-type strains and employing the fluorescence stain test revealed actin-rich pedestals,Keratin,Tublin under adherent O157: H7Δz3672 were indistinguishable from those formed by the wild-type strains, indicating thatΔz3672 is not required for this activity in vitro.In FAS(fluorescence actin staining) assay, we found an interesting phenomenon: the ability of induce actin polymerization may enhance if O157:H7 is cultured in DMEM (10% FBS) rather than in LB. After cultured in different media conditions (LB, DMEM, DMEM containing 10% FBS, DMEM containing 25m mol/L HEPES), O157: H7 were test in the following aspects: the growth rates, the ability of adhesion to HeLa cells, actin accumulation and the expression of proteins. O157: H7 grew slowest when cultured in DMEM containing 10% FBS, but the adhesion to HeLa cells and actin accumulation is the strongest. Attaching and effacing (A/E) lesions are characterized by the localized destruction (effacement) of intestinal epithelial microvilli, an intimate attachment between the bacterium and the host cell apical membrane, and the formation of pedestal-like structures containing high concentrations of actin and intermediate filaments directly beneath sites of bacterial attachment. Thus, it may enhance the ability of A/E lesion for O157: H7 when cultured in DMEM (10% FBS).In this study, we constructed O157: H7Δz3672 strain, and illuminated z3672 deletion mutant strain can increase the expression level of outer membrane protein A. It is a basal study for further research of Z3672 protein and FlxA-like protein family. O157: H7 may do more damage to infected HeLa cell in vitro if cultured in DMEM (10% FBS). O157: H7 may respond properly to surrounding environment (containing FBS) to corordinate virulence gene expression, but further study is still needed to understand the pathogenic mechanism.