Excavation And Study Of Oxidative Stress Related Genes In Extraintestinal Pathogenic Escherichia Coli

Extraintestinal Pathogenic Escherichia coli?ExPEC?is a kind of important zoonosis,which can cause the extraintestinal tissue infection of human and animal,resulting in huge economic losses in the livestock industry and serious harm to human health.When ExPEC infects the host,it will be resisted by the immune mechanism in the host.For example,phagocytes release a large amount of reactive oxygen species to kill the bacteria,which indicates it is necessary to identify the genes related to oxidative stress.We constructed a library of ExPEC mutants,screened for the mutants sensitive to hydrogen peroxide,identified the corresponding genes of mutants,and further studied their effects on bacterial survival and pathogenesis.The following results were obtained:1.Screening of genes related to the resistance of Extraintestinal Pathogenic Escherichia coli to oxygen stressWe first constructed 6,500 transposon inserted mutant strains,and then screened the growth characteristics of mutant strains with treatment of LB containing H₂O₂,and obtained 13 mutant strains with significantly reduced oxygen resistance.Tail-PCR was used to locate the inserted gene.According to the comparison of bacterial competitiveness and biofilm formation ability of mutants,four genes were selected for knockout to construct gene deletion strains.Comparing colonization of tissues in mice and abilities of bacterial adhesion and invasion,mutant N-2 was used for further analysis.The gene has only been annotated as a polymerase and contained the Wzy_C superfamily domain.We named it pcw and constructed its complement C-pcw.2.The absence of pcw affected the adaptability to peroxide environmentBy measuring the growth curve of RS218,?pcw and C-pcw in oxygen pressure,we found that deletion of pcw resulted in high susceptibility to H₂O₂,compared with WT and C-pcw.In order to explore the reason of slow growth in oxygen environment,we measured levels of WT,?pcw and C-pcw intracellular reactive oxygen species?ROS?under the stimulation of oxygen.Our results indicated that intracellular ROS level of?pcw was significantly more than intracellular ROS level of WT and C-pcw.The above results can prove that the absence of pcw affected the adaptation to peroxide environment.3.Deletion of pcw resulted in the loss of O antigen,which affected the synthesis of LPSIn order to verify the O antigen enzyme activity of Pcw,we compared the LPS structure of WT,?pcw and C-pcw,by silver stain and Western Bolt experiment.As a result,the LPS structure of the WT strain and C-pcw presented a typical ladder-like banding pattern.However,the LPS ladder of?pcw was almost absent.4.pcw was involved in the pathogenicity of E.coliMononuclear macrophage of mice?RAW264.7?and human brain microvascular endothelial cells?HBMEC?as the cell model,the resistance of bacteria to phagocytosis and intracellular survival after phagocytosis and adhesion and invasion abilities were compared.As a result,deletion of pcw caused decreased resistance to the phagocytosis of RAW264.7,reduced the intracellular survival of bacteria in RAW264.7,and reduced E.coli adhesion and invasion of HBMECs.Compareing the tissue colonization WT,?pcw and C-pcw,indicating?pcw has a lower tissue colonization capacity.After the mice were challenged with the same amount of bacteria,it was found that the survival rate of the mice in which?pcw was injected was significantly higher than that of WT and C-pcw.