The Characteristics Of OmpT Deletion Strain In Avian Pathogenic Escherichia Coli And Its Effect On The Expression Of Chicken Small Intestinal Inflammation Related Genes

The avian pathogenic Escherichia coli disease caused by Avian pathogenic Escherichia coli?APEC?is recognized as the major bacterial disease affecting the world poultry industry and it brings significant economic losses to the poultry industry.APEC's virulence factors are pilus,iron intake system,toxins,invading enzymes and outer membrane proteins,etc.The pathogenicity is complex and the serotypes are diverse.There are many ways to regulate the gene transcription by bacteria,and the PhoP/Q binary regulatory system is one of the key pathways.Among them,the expression of genes related to regulation and outer membrane protein synthesis is one of its functions.Our previous study found that the loss of APEC phoP/Q caused a significant down regulation of ompT gene expression.Outer-membrane protease T?OmpT?is an outer membrane protease in Gram-negative bacteria.It is not only acts as a protease for hydrolyzed proteins,but also may be a virulence-related factor of bacteria.Pathogenic bacteria play a role in the pathogenesis of infection.In this study,the ompT deletion strain and complementation strain were constructed by Red recombination technology,and their biological characteristics were analyzed.The deletion strains were sequenced and analyzed for differentially expressed genes based on RNA-Seq technology to study the effect of ompT gene deletion on bacterial virulence.To provide further theoretical basis for the further study of the pathogenic role of ompT gene in avian pathogenic E.coli.The research contents and results are as follows:1.Construction of ompT deletion and complementation strains and analysis of their biological characteristics and pathogenicityIn this experiment,AE17 was used as a wild strain,and the ompT deletion strain and complementation strain were successfully constructed by Red recombination technology.Bacterial growth curve measurements showed no significant difference in the ompT gene-deleted strains compared to wild-type strains.Environmental tolerance tests showed that the ompT gene deletion resulted in a significant increase in acid resistance?p<0.01?,a significant decrease in alkali resistance?p<0.05?,and a significant decrease in heat resistance?p<0.01?.The LD₅₀0 assay showed that the ompT deletion strain had reduced virulence in chicks.The results of the tissue-borne bacteria test showed that the amount of bacteria in ompT-deficient tissue?spleen,liver,lung?decreased.The results of exercise test,drug susceptibility test and chicken macrophage phagocytosis test showed that compared with the wild strain,the ompT gene deletion showed no significant difference in the APEC exercise ability,antibiotic sensitivity and anti-phagocytic ability.2.Screening for differentially expressed virulence genes in APEC ompT deletion based on RNA-SeqTo explore the effect of ompT deletion on APEC gene levels,differently expressed genes resulting from ompT deletion were screened from the level of transcription using RNA-Seq technology.The results showed 490 differently expressed genes,of which 125were down-regulated and 365 were up-regulated.The results of GO function indicated that the differential genes mainly involved in the biological processes such as iron homeostasis,lyase activity,peptidoglycan biosynthetic process,and regulation of cell shape,etc;the results of KEGG pathway analysis mainly involved bacterial chemotaxis,biofilm formation,and flagellar assembly,lipopolysaccharide biosynthesis pathways,etc.The virulence-related genes such as fliY,ompF,ibeT,aphA,motB,rfaQ,flgK,cheA,irp2,asnC,ycgR,lpxK,kpsU were selected.The expression of some of the differential genes?fliY,ompF,ibeT,aphA,motB,rfaQ,cheA and irp2?was verified by RT-PCR.The results were consistent with the trend of gene expression in RNA-Seq,indicating that the results of RNA-Seq were reliable.3.Inflammation related genes mRNA transcription levels in the chicks of intestinal tissue after infected with APEC and its ompT deletion strain by RT-PCRThe expression levels of PTGDS and MME mRNA in the jejunal inflammatory tract of chicks were compared by RT-PCR.The results showed that the expression level of PTGDS gene mRNA in the challenge group was significantly lower than that in the AE17challenge group at 12h,24h and 48h after challenge?p<0.01 or p<0.05?,and the expression of PTGDS gene mRNA at 12h and 24h after challenge was observed.The amount is higher than 0h,48h and 72h.After 12 hours of challenge,the expression of MME gene mRNA in all challenge groups was significantly lower than that in 0h;the expression levels of MME mRNA in the challenge group were significantly lower than those in the AE17 challenge group at 24h and 48h after challenge with the virus?p<0.01or p<0.05?.The above results indicate that the ompT deletion strain and complementation strains of Avian pathogenic E.coli strain were successfully constructed in this study.OmpT deficiency resulted in enhanced acid resistance,decreased alkali-tolerance,heat resistance,reduced bacterial load in the liver,spleen,and lungs,significantly reducing the toxicity and pathogenicity of APEC.But no significant effect on exercise,drug sensitivity,anti-phagocytic ability.Deletion of the APEC ompT gene caused changes in the expression of virulence related genes?fliY,ompF,ibeT,aphA,motB,rfaQ,flgK,cheA,irp2,asnC,,ycgR,lpxK,kpsU?.The expression of PTGDS and MME mRNA of jejunum was increased in chickens infected with APEC,and the expression of PTGDS and MME mRNA of jejunum was affected after the ompT was deleted,indicating that the inflammation related genes PTGDS and MME participated in the process of resisting APEC infection.ompT may participate in the APEC infection process.