Construction And Biological Characteristics Of UhpA Gene Deletion Strain Of Edwardsiella Piscicida

Edwardsiella piscicida?E.piscicida?is a Gram-negative,rod-shaped,motile bacterium belonging to the family of Enterobacteriaceae.E.piscicida is a conditional pathogen.It widely distributes in aquatic environment and can infect a large number of fish species,which leads to a systemic disease called Edwardsiellosis and causes enormous economic losses around the world.The study found,Edwardsiella piscicida?E.piscicida?is an invasive intestinal bacterium.fucose signals were found to regulate two-component system of FusKR which could promote virulence of other bacteria,especially type III secretion system?T3SS?.Related studies have confirmed that 6-phosphate glucose regulates the uhpA/uhpB two-component system and further regulates other virulence genes,but the uhpA gene has no clear understanding of fucose-activated FusKR-related genes and T3SS virulence genes and their mechanisms.Therefore,we construct a gene uhpA deletion mutant of E.piscicida named?uhpA,subsequently identify phenotypic analysis and the function of gene uhpA using compare to the wild type strain E.piscicida EIB202.We used homologous recombination in the construction of the uhpA gene deletion strain?uhpA of the E.piscicida EIB202,and then compared it with the wild strain.The study found that glucose 6-phosphate significantly increased the growth rate of wild strain EIB202 of E.piscicida?P<0.05?;wild strain EIB202 supplemented with glucose 6-phosphate as a nutrient source added glucose 6-phosphate as a nutrient source The wild type and the deletion strain of the deletion strain and the glucose-free glucose-phosphate as the nutrient source showed significant growth?P<0.05?.These results suggest that the gene uhpA deletion in E.piscicida affect G-6-P usage.According to the references,it is speculated that uhpA gene may affect fusKR-related genes and T3SS virulence genes activated by fucose.Therefore,we investigated the effects of fucose and uhpA genes on FusKR-related genes and T3SS virulence genes of E.piscicida.The results showed that the mRNA levels of FusKR genes,a permease?fucP?gene,a phosphate aldolase?fucA?gene and a regulator?fucR?gene in EIB202 cultured with L-fucose were significant higher than without L-fucose added?P<0.05?,especially with 30 mM L-fucose added.In addition,at a concentration of L-fucose of 30 mM,The results demonstrated that the mRNA levels of gene fucP,fucA and fucR in?uhpA were 1.65-fold,2.12-fold and 1.82-fold upper than EIB202?P<0.05?.The results demonstrated that the mRNA levels of gene T3SS?esrB?esrC??Flagellar gene?fliC?flgN??Hemolysin gene?EthA?EthB??T6SS?EvpB?EvpC?in?uhpA were 1.27-fold,1.13-fold,1.51-fold,1.21-fold,0.80-fold,0.72-fold,1.28-fold,1.23-fold upper than EIB202?P<0.05?.The results indicate that the uhpA gene can influence the fucose signaling to regulate key genes in the FusKR system,which in turn affects the expression of T3SS and other virulence-related genes.E.piscicida?uhpA exhibited an increase in virulence compared to that of E.piscicida EIB202[LD₅₀0 value:?2×10⁴ CFU/fish?and LD₅₀0 value:?1×10⁵ CFU/fish?respectively].It is fully indicated that the pathogenicity of E.piscicidais caused by the deletion of the uhpA gene.Cytokines play a very important role in the fight against pathogens in fish.We determined the effect of the absence of the uhpA gene on the induction of cytokines in zebrafish.Fish infected with EIB202 and?uhpA showed significantly higher?P<0.05?gene expression of IL-1??TNF-??INF-??TGF-?in spleens than fish treated with PBS in the whole observed period.Fish infected with?uhpA showed significantly higher?P<0.05?gene expression of IL-1??TNF-??INF-??TGF-?in spleen than fish treated with EIB202 in the whole observed period.The results of bacterial infection of mouse macrophages showed that the expression of IL-1?and IL-6 was significantly increased in the early stage of infection with E.piscicida?0-12h?.At 6h,the cells of IL-6 in the?uhpA treatment group were treated.The transcription level of the factor gene was significantly higher than that of the EIB202group?P<0.05?.At 6h and 12h,the transcription level of cytokine IL-1?gene was significantly higher than that of EIB202 group?P<0.05?.In conclusion,this study successfully constructed the uhpA deletion strain of E.piscicida.The deletion of uhpA gene enhances its pathogenicity and changes its mode of inducing cytokine production by zebrafish and macrophages.This study provides a research basis for the pathogenesis of E.piscicida and the prevention and control technology of Edwardsiellosis.