Study On The Stress Resistance And Pathogenicity Of HutZ Of Edwardsiella Piscicida And Its Regulated Mechanism

Edwardsiella piscicida,was previously called E.tarda,is an important pathogen in mariculture,which can infect a variety of important economic aquatic animals and cause disease,such as Paralichthys olivaceus,Scophthalmus maximus and Oreochroms mossambcus.Currently,E.piscicida seriously restricts the healthy development of aquaculture in China.So far,the pathogenic mechanism of E.piscicida has not been fully understood.Iron is indispensable for E.piscicida to cause host disease,however,There is very little free iron in the host,mainly exists in the form of hemoglobin,therefore,the heme uptake system,which can release iron ions using heme,plays a crucial role in the pathogenesis of E.piscicida.In previous studies of E.piscicida bacteroomics,it was found that a gene annotated with the hemoglobin utilization protein was likely involved in the pathogenicity of bacteria.To further explore and clarify the function of this gene,this research mainly take hemoglobin utilization protein HutZ in E.piscicida(name HutZEp)as the research object,purposed through the function of HutZEp and its interaction with Ferric uptake regulator(Fur)analysis,expounded the role of HutZEp in the process of resistance and pathogenicity of E.piscicida and its regulated mechanism,so as to provide an effective way for the prevention and treatment of E.piscicida.The main research contents and results are as follows:(i)Protein structure and function analysis of HutZEp.Bioinformatics analysis showed that HutZEp is a protein associated with heme utilization,which has the highest homology with the heme reductase ChuY in E.coli(50.23%).hutZEp is co-transcribed with two upstream heme-related genes hutW and hutX,and the three together form an operon hutWXZ.The protein function of HutZEp was analyzed by heme binding test and protein enzyme activity test,it was found that HutZEp cannot bind to heme,nor can it use NADPH and NADH as electron donors to reduce FMN.Based on this,we speculate that HutZEp is not required for heme utilization by E.piscicida.(ii)Analysis of the role of hutZEp in E.piscicida adversity adaptation and pathogenicity.A markerless hutZEp in-frame mutant strain,TX01?hutZ,was constructed by using gene knockout.The adversity test showed that the deletion of hutZEp did not significantly affect bacterial growth in normal medium,in iron-deficient conditions,or in the presence of haem,but significantly retarded the ability of bacterial biofilm growth and athletic;RT-qPCR analysis showed that the expression of known genes related to biofilm growth was not affected by hutZEp deletion,which indicated that HutZEp was probably a novel factor promoting biofilm formation in E.piscicida;Compared to the wild-type TX01,TX01?hutZ exhibited markedly compromised tolerance to acid stress and host serum stress.Pathogenicity analysis showed that deletion of hutZEp significantly impaired the invasion and intracellular reproduction ability of E.piscicida,and significantly reduced the ability to infect host tissues.In contrast to the wild strain TX01,the mutant TX01?hutZ was defective in blocking host macrophage activation.The complementary strain TX01?/hutZC of the mutant TX01?hutZ is comparable to the wild strain TX01 in terms of bacterial resistance and pathogenicity.Therefore,HutZEp is not only an important virulence factor for E.piscicida,but also a new biofilm formation factor for E.piscicida,which plays a vital role in the process of bacterial resistance and pathogenicity.(iii)Study on the regulated mechanism of hutZEp.The hutZEp promoter was predicted,and the hutZEp promoter p283 sequence was obtained by PCR,and it was ligated with the reporter vector pSCll vector to obtain the recombinant plasmid pSZ283,which was transformed into E.coli DH5?.By X-Gal plate detection,p283 had promoter activity.pSZ283 was cotransformed with expression vector pT3 and fur overexpression vector pTFur into DH5a competent cells to obtain recombinant strains DH5?/pSZ283-pT and DH5?/pSZ283-pTFur.The assay of promoter activity showed that the ?-galactosidase activity of DH5?/pSZ283-pT was 95 times higher that of DH5?/pSZ283-pTFur,It is shown that Fur has a strong inhibitory effect on hutZEp promoter p283.In order to further clarify the effect of Fur on promoter p283,Fur protein was expressed and purified in vitro.Gel retardation test(EMSA)found that Fur protein can bind to hutZEp promoter p283.RT-qPCR showed that the expression of hutZEp in the fur mutant strain TX01?fur strain was 145 times that of the wild strain TX01;Western blot analysis showed that the expression of HutZEp in TX01?fur strain was significantly higher than that in TX01;EMSA experiments showed that Fur protein was able to bind to hutZEp promoter p283.These results fully demonstrate that,hutZEp is directly negatively regulated by Fur protein.In summary,HutZEp may be a new bio film-forming factor that plays an important role in the process of E.piscicida biofilm formation.At the same time,HutZEp plays a role in resisting to acid and other adversity environments.Moreover,HutZEp is involved in anti-serum bactericidal effects and plays an important role in the process of pathogens infecting host cells and tissues.In addition,this research also found that although HutZEp is not required for bacterial heme utilization,the expression of HutZEp is directly and negatively regulated by Fur,an iron uptake regulator.This experiment is the first study of the function and regulated mechanism of HutZEp in E.piscicida,from a new entry point,so as to provide a new theoretical basis for the prevention and the control of E.piscicida.