Biological Characteristics Of Plasmid PEIB202 In Edwardsiella Piscicida

Edwardsiella piscicida（E.piscicida）infects fish and seriously threatens the healthy development of aquaculture,causing clinical symptoms of abdominal swelling,fins,abdomen and surface hemorrhage,resulting in the death of aquaculture fish in huge loss batches.In addition to being powerful pathogenic,E.piscicida is also a communicator of spread resistance genes into the breeding environment.The resistance gene carried by itself is transmitted to other pathogenic bacteria in the environment,resulting in new drug-resistant strains,which brings certain difficulties to farmers in the prevention and control of drugs.Studies have shown that E.piscicida carries a 43.7 kb mobile conjugation plasmid,which carries a variety of resistance genes,and another study found that the deletion of fucR,a key gene of the fucose operon,has the greatest impact on type Ⅳ secretion,tetracycline resistance genes,chloramphenicol resistance genes and some transposase genes.It has convinced that the genes with greater impact after the deletion of fucR gene are mainly found in the plasmid of E.piscicida superior.However,the function and mechanism of action of plasmids are not fully known by current research.This experiment mainly analyzes the biological characteristics of plasmid pEIB202 in E.piscicida.In this study,a plasmid deletion strainΔpEIB202 of E.piscicida was constructed by the principle of homologous recombination to explore the relationship between the type Ⅳ secretion system related genes on the plasmid and the and the effect of conjugation on bacteria,and we construct the double deletion strainΔPR to explore the relationship between the gene fucR and the plasmid.Finally,we compare the difference among the constructed deletion strainsΔpEIB202 andΔPR with laboratory-preserved strainΔfucR and wild strain EIB202 for a series of biological characteristics comparison analysis.The results of bacterial growth,motility conjugation tests showed that except lacked tetracycline and chloramphenicol resistance it had no significant difference of the growth rate and motility amongΔpEIB202,ΔPR and the wild strain EIB202,indicating that the plasmid pEIB202 is the cause of bacterial resistance.The results of conjugation ability showed that the ability of bacteria to receive foreign plasmids was weakened after plasmid deletion.However,after the deletion of the fucR,which is directly related to the plasmid,the ability of bacteria to transmit plasmids and receive foreign plasmids is weakened.The Ⅳ secretion system genes carried on the plasmids of E.piscicida are directly related to the conjugation of bacteria.Therefore,the effects of fucR gene and pEIB202 plasmid deletion on the transcription of T4SS-related genes were determined.The results showed that after the deletion of the fucR,genes related to the inner membrane complex（IMC）（virB3,virB6,virB8,virB10）,related to conjugative fimbriae（virB2）,and ATPase synthesis related genes（virD4,virB4,virB11）0.25-fold,0.30-fold,0.42-fold,0.71-fold,0.15-fold,0.73-fold,0.25-fold and 0.56-fold（P<0.05）.Respectively,the transcript levels of the gene virB9 associated with the outer membrane core complex（OMCC）.It was 0.82-fold compared with the wild strain（P>0.05）.The results showed that the fucR gene could affect the key genes of T4SS,which in turn affected the conjugation ability of bacteria.It shows that the plasmid pEIB202 is the vector of E.piscicida for conjugation,and fucR can regulate the conjugation ability of bacteria by regulating the genes related to the conjugation on the plasmid.Trough assignment the median lethal dose LD₅₀of each mutant,the LD₅₀values of EIB202,ΔfucR,ΔPR andΔpEIB202 were 1.73×10~4CFU/fish,1.2×10~5CFU/fish,5×10~4CFU/fish and 8.3×10~4CFU/fish.The results showed that the deletion of fucR and the deletion of plasmid pEIB202 reduced the pathogenicity of E.piscicida to varying degrees,but the effect was not significant.Several virulence-related genes inΔpEIB202 were detected by qPCR,and several key T3SS genes（esrB,esrC）,flagellar genes（fliC,flgN）,hemolysin genes（EthA,EthB）,and T6SS key genes（EvpB,EvpC）.The mRNA transcription levels of the virulence-related genes were 1.61-fold,0.93-fold,1.16-fold,0.84-fold,0.38-fold,0.57-fold,1.61-fold and 1.91-fold than those of the wild strain（P<0.05）.Besides,we admeasurement the colonization capacity of EIB202,ΔpEIB202 andΔPR in the zebrafish.In general,E.piscicida colonization mainly occurred within 12 h after challenge,and the colonization amount decreased significantly after 12 h.The colonization ability of different mutant strains was compared and analyzed,and it was found that the colonization amount ofΔpEIB202 in the zebrafish gut was significantly lower than that of EIB202 before 48 h（P<0.01）,and almost can not findΔpEIB202 bacteria could be detected in the gut after 48 h.After 72 h,there was no significant difference betweenΔpEIB202 and the wild strain（P>0.05）,and the colonization ofΔPR was similar to that ofΔpEIB202.The surface lipopolysaccharide structure of Gram-negative bacteria plays an important role in protecting bacteria from external damage and bacterial pathogenicity.Fluorescence PCR detection of lipopolysaccharide synthesis genes ofΔpEIB202 showed that the transcription levels of genes（rfaD,rfaQ,rfaC）related to LPS core oligosaccharide synthesis were 0.7-fold,0.83-fold,and0.39-fold than those of the wild strain（P<0.05）.Genes related to lipid A synthesis（lpxD,lpxB）were 0.9-fold and 1.2-fold than those of wild strains（P>0.05）.Based on the above experimental results,the plasmid pEIB202 can increase the colonization of E.piscicida in the zebrafish intestine,enhance the pathogenicity of E.piscicida.And at the same time,it can affect the key genes of LPS synthesis and enhance the toxicity.In conclusion,in theΔpEIB202 andΔPR deletion strains successfully constructed in thisexperiment,it was found that the deletion of plasmid pEIB202 had no significant effect on the growth and virulence of bacteria,but significantly reduced the colonization ability of the bacteria and the conjugation ability of the strains.The research on strainΔPR found that its conjugation ability and the changing trend of toxicity were similar toΔpEIB202,which further proved the function of plasmid pEIB202.The study ofΔfucR further confirmed that the fucR gene can reduce the spread of bacterial resistance plasmids by affecting related genes on the type Ⅳ secretion system.This experiment studied the pathogenic mechanism of E.piscicida plasmid pEIB202,provided new ideas and theories for the development of E.piscicida disease prevention and control technology,and had important research significance.