The Construction Of OxyR Gene Deletion Strain And The Functional Identification Of OxyR Gene In Haemophilus Parasuis

Haemophilus parasuis?H.parasuis?is a case of multiple serositis and arthritis in pigs caused by H.parasuis.Over the years,the epidemic of this disease has brought serious economic losses to the pig industry in China as well as in the world.The OxyR regulatory protein belongs to the LysR family of transcriptional regulators and plays a key role in the antioxidant defense system of the bacteria.This regulator has also been considered to be related to the regulation of various bacterial virulence factors.Up to now,there have been few reports on the antioxidation of H.parasuis,and there has been no report on the oxyR gene in H.parasuis.Based on the whole genome sequencing of H.parasuis serotype 11SC1401 strain in our laboratory,the oxyR gene deletion strain?H.parasuis SC1401?oxyR?and complementary strain?H.parasuis SC1401?oxyR pLS88::oxyR?were constructed,and analyzed for its biological phenotypes.By the global comparative transcriptomics and proteomics analysis,there are several important genes and important proteins which are related to oxidative stress of H.parasuis were identified.These studies provide scientific basis for elucidating the role of oxyR in the pathogenesis of H.parasuis and the molecular regulation mechanism of anti-oxidative stress,and provide new valuable information for the prevention and control of the disease.1.Construction of oxyR gene deletion strain of H.parasuisThe H.parasuis strain SC1401 was used as the parent strain,and the oxyR gene upper arm fragment?U?,Kan gene fragment?K?and oxyR gene lower arm fragment?D?were fused by fusion PCR to obtain fragment?UKD?;UKD was inserted into pK18mobsacB vector.The recombinant vector UKD-pK18 was obtained;UKD-pK18 was transformed into H.parasuis SC1401 strain by natural transformation method.Single colony was picked on TSA medium containing kanamycin and identified by PCR and Western Blot,then the H.parasuis oxyR gene deletion strain was generated?H.parasuis SC1401?oxyR?.The complete open reading frame?ORF?of oxyR gene was amplified by PCR and complete oxyR fragment was obtained;the oxyR fragment was ligated with the shuttle plasmid pLS88 to obtain the complementary plasmid pLS88::oxyR,which was electroporated into the oxyR gene-deleted strain?H.parasuis SC1401?oxyR?;the complementary strain?H.parasuis SC1401?oxyR pLS88::oxyR?was generated and identified by PCR and Western Blot.2.Biological characteristics of oxyR gene deletion strain of H.parasuisFor H.parasuis SC1401,H.parasuis SC1401?oxyR,and H.parasuis SC1401?oxyR pLS88::oxyR were examined by Gram stain microscopy,and no significant differences were observed for the morphology of this cells;the results of growth curve measurement,the growth curve was measured at 12 h when for H.parasuis SC1401,H.parasuis SC1401?oxyR and H.parasuis SC1401?oxyR pLS88::oxyR,the OD600 was 1.68,0.31and 1.44 respectively,showing that the absence of oxyR resulted in significant growth defects of H.parasuis.The H.parasuis SC1401?oxyR pLS88::oxyR can restore to the normal growth.The oxidative stress resistance experiments showed that all the inhibition rings of H.parasuis SC1401?oxyR were greater than that of H.parasuis SC1401 and H.parasuis SC1401?oxyRpLS88::oxyR under different H₂O₂ concentrations,indicating that H.parasuis SC1401?oxyR is more sensitive to H₂O₂ than H.parasuis SC1401.The biofilm formation assay measured the amount of biofilm formation of H.parasuis SC1401?OD600,0.520?,H.parasuis SC1401?oxyR?OD600,0.245?and H.parasuis SC1401?oxyR pLS88::oxyR?OD600,0.466?suggested that the oxyR gene had an effect on the biofilm formation of H.parasuis.Histopathological analysis of challenging mice,found that the parental strains in the lung,liver,brain,spleen formed obvious symptoms of different lesions,however,the oxyR deleted strains in all tissue were significantly reduced or even no lesions were found,resulted that the oxyR is associated with the virulence of H.parasuis.3.Transcriptomic Comparasion between H.parasuis SC1401 and oxyR gene deleted StrainTranscriptomic comparasion of H.parasuis SC1401 and H.parasuis SC1401?oxyR was performed using RNA-seq technology.A total of 466 differentially expressed genes?DEGs?were found in our study,of which 240 genes were up-regulated and 226 genes were down-regulated.The DEGs accounted 20.86%of the whole genome for H.parasuis SC1401.The functions of genes of DEGs mainly include transcriptional regulation,DNA replication,recombination and repair,and oxidative stress.GO annotations of DEGs showed that there are mainly organic cyclic compound biosynthetic process,heterocycle biosynthetic process,aromatic compound biosynthetic process and oxidation-reduction process.The KEGG pathway is mainly composed of Biosynthesis of secondary metabolites,Microbial metabolism in diverse environments,ABC transporters,and Carbon metabolism.4.Proteomics comparasion of H.parasuis SC1401 and oxyR Gene Deletion StrainsA comparative proteomic analysis of the H.parasuis SC1401 isolated strains and the oxyR gene-deleted strains were performed using the iTRAQ quantitative proteome sequencing analysis technique.A total of 45 differentially expressed proteins were identified following the deletion mutation in the oxyR gene of H.parasuis.Among them,14 proteins were up-regulated,mainly lysogeny proteins HflD,ABC transporter substrate binding protein and DNA recombinant repair protein RecA,etc.;down-regulated by 31proteins,mainly ABC transporter permease,nitrate reductase catalytic subunit and iron sulfur Cluster scaffold protein IscU,etc.The GO enriched and KEGG enrichment analysis of the identified differentially expressed proteins was found to be mainly related to carbohydrate metabolism,TCA cycle,cell signal transduction,and bacterial pathogenicity.A comparative analysis of the transcriptomics and proteomics revealed that the Pearson correlation coefficient,0.261 was low.The corresponding gene functions were mainly carbon metabolism,homologous recombination and ABC transporters.In summary,the oxyR-deleted strain of H.parasuis was constructed for the first time in this study,and the oxyR gene and its associated pathogenic mechanisms were systematically studied.In particular,a wide range of differentially expressed genes and differentially expressed proteins were discovered from transcriptomic and proteomic levels.These studies provide a scientific basis and valuable information for further explanation and changes in the biological characteristics of oxyR gene deletion strains of H.parasuis,as well as for the study of oxyR function and molecular regulation mechanisms.