Biological Function Analysis Of FucP Protein Of Edwardsiella Tarda

Edwardsiella tarda（E.tarda）is an important zoonotic pathogen,which particularly infect aquatic animals.Studies show that the intestine is the main route of entry for E.tarda,suggest that colonization of E.tarda may be related to nutrient source in intestine or intestinal epithelial cells.In addition,fucose signals were found to regulate two-component system of FusKR which could promote virulence of other bacteria,especially type III secretion system（T3SS）.Therefore,we construct a gene fucP deletion mutant of E.tarda namedΔfucP,subsequently identify phenotypic analysis and the function of gene fucP using compare to the wild type strain E.tarda EIB202.In this study,fucP gene deletion mutantΔfucP was successfully constructed by homologous recombination method,and a series of comparative studies with wild strains were conducted.The results showed that EIB202 andΔfucP strains were translucent colonies on TSB agar and appeared as black-centered strains on SS agar,indicating that gene deletion has little effect on their basic biological characteristics.L-fucose-affected wild-type strains and mutant growth experiments that both two strains demonstrated significant increases in growth in response to the availability of L-fucose as a nutrient source（P<0.05）.Meanwhile,E.tarda EIB202 demonstrated significant increases in growth than fucP gene deletion when grown inα-MEM both with and without L-fucose.However,theΔfucP strain demonstrated significant growth faster inα-MEM with L-fucose than both two strains grown inα-MEM without L-fucose.Which suggest that the gene fucP deletion in E.tarda did not affect L-fucose using.Whereas,wild-type strains and mutant strains infecting zebrafish for determination of their median lethal dose(LD₅₀)results showed that the gene fucP deletion decrease the virulence of E.tarda,ΔfucP exhibited a decrease in median lethal dose(LD₅₀)in zebrafish[LD₅₀0 value:（6×10⁵CFU/fish）compared to that of EIB202 LD₅₀0 value:（3.4×10⁴CFU/fish）].In addition,the gene fucP deletion decrease the motility and colonization of E.tarda.We used wild strains and mutant strains to penetrate mucin and simulated intestinal test results showed that the mean total number of EIB202 significant higher thanΔfucP with or without L-fucose added.The number of E.tarda in anterior intestine of tilapia infected with EIB202 was significantly higher than infected withΔfucP after 6 and 12 h.The number of E.tarda in middle and posterior intestines of tilapia infected with EIB202 was significantly higher than infected withΔfucP after infection from 6 to 48 h.The total number of E.tarda intestine of tilapia infected with EIB202 was significantly higher than infected withΔfucP after infection 6 and 12 h.Regardless,evidence strongly suggests that the motility and colonization of E.tarda is activated by fucose.Which predict that fucose signals would activate the key genes of FusKR and T3SS.The results showed that the mRNA levels of FusKR genes,a permease（fucP）gene,a kinase（fucK）gene and a regulator（fucR）gene in EIB202 cultured with L-fucose were significant higher than without L-fucose added（P<0.01）,especially with 30 mM L-fucose added.In addition,the results demonstrated that the mRNA levels of T3SS genes,effector protein genes（eseB、eseC、eseD、eseE、eseG）,apparatus protein genes（esaB、esaD、esaK、esaL）,regulation protein genes（esrA、esr B、esrC）,chaperones protein genes（escA、escB）were all significant higher than without L-fucose,especially with 30 mM L-fucose added too.The results demonstrated that the mRNA levels of gene fucP,fucK and fucR inΔfucP were 0-fold,0.48-fold and 0.64-fold lower than EIB202（P<0.05）.In addition,the results demonstrated that the mRNA levels of the key genes of T3SS inΔfucP were from 0.37-fold to 0.65-fold lower than EIB202（P<0.05）.Cytokines are the key immune factors for fish against bacterial infections.We measured the effect of fucP gene deletion on the induction of tilapia cytokines.Fish infected with EIB202 andΔfucP showed significantly higher（P<0.05）gene expression of IL-1β、TNF-α、IFN-γ、TGF-βand HSP-70 in head kidney than fish treated with PBS in the whole observed period.Fish infected with EIB202 showed significantly higher（P<0.05）gene expression of TGF-βin head kidney than fish treated withΔfucP in the whole observed period.Fish infected with EIB202 andΔfucP showed significantly higher（P<0.05）gene expression of all cytokine including IL-1β,TNF-α,IFN-γ,cas-3,TGF-βand HSP-70 in spleen than fish treated with PBS in the whole observed period.Fish infected with EIB202 showed significantly higher（P<0.05）gene expression of IL-1β,TNF-αand TGF-βin spleen than fish treated withΔfucP in the whole observed period.The induced expression of cytokine in head kidney and spleen in fish demonstrated the activity and a local immune response of ex vivo anterior sac.In conclusion,the deletion of the fucP gene from E.tarda has no significant effect on its colony characteristics,but it significantly reduces the pathogenic factors such as the slowness of the E.tarda movement ability,colonization ability,and the ability to induce host immune responses.Further studies on the deletion of the fucP gene from E.tarda confirmed that fucose signalling modulates the transcription and expression of fucP,a key protein of FusKR,and further activates the transcription and expression of T3SS and enhances the ability to delay the colonization of E.tarda.This study investigated the pathogenic mechanism of the delayed E.tarda fucP protein and provided new ideas and theories for the prevention and control of the development of E.tarda disease.It has important research significance.