| Vibrio cholerae is a Gram-negative pathogen that can cause a devasating intestinal infectious disease,cholera.It is highly resistance to mutilple antibiotics which still a severe threaten for human life.The MarR(multi-drug resistance)family proteins are involved in the processes of antibiotic resistance,oxidative stress response,virulence activation and aromatic compounds catabolism.Therefore,studying on the physiological function of MarR-like proteins and the molecular regulation mechanisms of antibiotic resistance in Vibrio cholerae would provide new insights to the prevention and treatment of cholera and other infectious disease.In this thesis,based on the MarR HTH conserved motif,ten MarR-like proteins were indentified in genome of Vibrio cholerae N16961,among which the unknown functional proteins included VCA0955,VC0649,VC0493,VCA1068,VCA1058,VCA0889 and VC2103.In order to explore the physiological functions of these proteins in Vibrio cholerae,seven single gene in-frame deletion mutants were constructed and the diverenges between wild strain in biofilm formation ability,motility,antibiotic sensitivity,oxidative stress resistance and host colonization ability were detected:(1)Biofilm formation ofΔvca1058 andΔvca1068 mutants increased when 0.2% glycerol was used as carbon souces.Biofilm formation ofΔvca0955 mutants decreased when 0.2% glycerol was used as carbon sources while increased when 0.5% casein was used as carbon souces.The migration distance of seven single gene mutants was similar to that of wild strain in 0.3% LB soft plate.(2)Under the treatment of redox active antibiotic furazolidone and cumene hydroperoxide,Δvca1058 mutant were more sensitive than wild strain.Under the treatment of hydrogen peroxide and cumene hydroperoxide,Δvca0955 mutant were more sensitive than wild strain.(3)In the host colonization assay,the competitive colonization ability ofΔvca0955,Δvca1058,Δvca0889 andΔvc2103 mutants in the intestinal tract of adult mice decreased by 8-10 times compared to wild strain.Due to the strong resistance of Vibrio cholerae to furazolidone and which the resistance mechanism was not cleared,the furzaolidone sensitive phenotype ofΔvca1058 mutant was further studied.The complement of vca1058 could restore the furazolidone resistance inΔvca1058 mutant.According to the typical transcriptional regulation mode of MarR-like proteins,it is suggested that the adiacent reverse gene vca1057 is the target gene of VCA1058.The sensitivity ofΔvca1057Δvca1058 double genes deletion mutants,wild strain and E.coil DH5α to fuazolidone was depended on the expression of vca1057 intracellular,which suggested that VCA1057 is a sensitizing agent of furazolidone.Through luminescence detection of reporter plasmid with vca1058 promoter,it was revealed that VCA1058 represses its own gene expression.Meanwhile the increased expression of intracellular VCA1058 leads to a dercease in the transcriptional level of gene vca1057 in a dose-dependent manner.It is indicated that short chain dehydrogenase VCA1057 could enhance the intracellular activity of furazolidone,and the MarR-like protein VCA1058 regulated the sensitivity of bacteria to furazolidone through the expression of transcriptional regulation gene vca1057.There exists several MarR-like proteins in Vibrio cholerae.Due to the conservativeness structure in the N-terminus and the variability structure in the C-terminus of MarR-like proteins,it is speculated that the regulation at gene level is complex and the function is complementary,which lead to the inability to detect phenotypes under the condition of single gene deletion.Therefore,seven MarR proteins gene deletion mutant,Δ7marR,was constructed in Vibrio cholerae C6706,and the host colonization ability was tested.Further the expression level of virulence gene tcpA was detected.Results showed that the competitive colonization ability ofΔ7marR mutant was 1000 times lower than that of wild strain in adult mice,while the competitive in infant mice and the tcpA expression showed no obvious divergence to wild strain.In conclusion,seven MarR-like proteins with unknown functions in Vibrio cholerae was discovered in this thesis and they could responed to environmental singal perturbations,such as nutrients,antibtiotics,oxidative stress,thus regulating the environmental adaptability and host pathogenicity of Vibrio cholerae. |
PDF Full Text Request