The Influence Of Transcriptional Regulator Fur On The Virulence Of Riemerella Anatipestifer

Riemerella anatipestifer infection is a disease to duck,goose,turkey and many waterfowl,which characterized by fibrinous pericarditis,perihepatitis and airsacculitis.This disease which had high morbidity and mortality was distributed widely in the word.It was defined as the second class of legal animal epidemic disease.As RA have complex serotypes,and effective cross protection among different serotypes is low,leading to the difficulty of disease prevention.Once this disease was occoured,it was difficult to elimate it completely.Now this disease was mainly controlled with medication.Thus,it is great significance to focus on the pathogenesis mechanism of RA which is helpful to the prevention of the occurrence of this diease.Iron is an essential element in virous metabolic pathways of bacteria.In addition,iron overload in the bacteria is associated with hydroxyl redical·OH produced by Fenton reaction,and it was toxic to the bacteria.The ferric uptake regulator protein(Fur)is a regulator of iron metabolism in gram-negative bacteria.It not only regulated iron acquirement and utilization,but also involved in many life activities such as the growth,metabolism and virulence.However,the role of Fur in the virulence of RA has not been demonstrated.Gene deletion was a basic method of molecular biology study.To date,there was not study of unmarked gene deletion system in RA.For this purpose,we constructed an unmarkd gene deletion system containing phe S which encode phenylalanine-t RNA transferase and indicated marker lac Z.Using this system,we constructed unmarked fur gene deletion mutant strain RA-YM?fur.Then the shuttle plasmid was constructed by adding replicon of plasmid p RA-JX.Using this shuttle plasmid,we contructed the complemented strain C?fur successfully.Though expreminet on ducklings,it was demonstrated that fur was a virulence factor of RA.Moreover,this study also performed the investigation of the gene-regulating function of Fur using RNA-seq.The research contents are summarized as follows:1.Construction of fur gene deletion mutant strain RA-YM?fur and the complemented strain C?furWe constructed suicide plasmid p RE-lac Z-mphe S-spc and shuttle plasmid p RES-JX-spc which were apply to establish markless gene deletion mutant strain and complemented strain in RA.The result of growth inhibition test showed that RA was sensitivity to 0.2% 4-chloro-DL-phenylalanine.So phe S can be used as counter-selectable marker in RA.To decline the probability of homologous recombination at this site,thephe S gene was engineered by substituting alternative base at numerous positions.The sequence similarity rate between wild type phe S and mphe S was 71%.In addition,we cloned the indicated marker lac Z which is driven by promoter rps L.With the indicated marker,we can distinguish the homologous recombination state by the colour of the strain.Finally,we constructed a new suicide plasmid p RE-lac Z-mphe S-spc using the skeleton of plasmid p RE112,with the counter-selectable marker phe S,indicated marker lac Z and spc gene.On the other hand,we also constructed the shuttle plasmid p RES-JX-spc by adding the replicon region of plasmid p RA-JX.With this shuttle plasmid,the complemented strain can be constructed successfully.Using the method,we constructed fur gene deletion mutant strain RA-YM?fur and the complemented strain C?fur.The specific operations are as shown below.The homologous Leftarm,Rightarm of fur gene were amplified respectively.Leftram and Rightarm were linked together using the method of overlap PCR,and then subcloned into plasmid p RE-lac Z-mphe S-spc.The E.coli X7213 strain harbored plasmid p RE-lac Z-mphe S-spc-fur was as the donor strain,and RA-YM was as the recipient strain,fur gene deletion mutant strain RA-YM?fur was constructed using two steps homologous recombination.The merodiploid was selected by spc firstly.Then the fur gene deletion mutant was selected by phe S and lac Z.Moreover,the promoter and CDS region of fur gene were amplified respectively.The promoter and CDS region of fur gene were linked together using the method of overlap PCR,and then subcloned into shuttle plasmid p RES-JX-spc to construct recombination shuttle vector p RES-JX-spc-fur.The E.coli X7213 strain transformed with plasmid p RES-JX-spc-fur was as the donor strain,and RA-YM?fur was as the recipient strain,the complemented strain C?fur was constructed using conjugational transfer.The virulence of RA-YM,RA-YM?fur and C?fur was measured by Challenge experiment of Cherry Valley Ducks.The results showed the the median lethal dose(LD50)value of RA-YM,RA-YM?fur and C?fur was 2.0×106 CFU,1.6×108 CFU,1.2×107 CFU respectively.The virulence of mutant strain RA-YM?fur was 80 times attenuated compared with that of RA-YM.And the virulence to ducks was partially restored when the mutant was complemented with the shuttle plasmid p RES-JX-spc-fur.The analysis of bacterial loading of target tissue showed that the bacterial loading was higher in RA-YM strain compared to RA-YM?fur strain at 24 h and 48 h.The pathological investigation demonstrated that the lesion of target tissue was more significant seriously in RA-YM strain compared to RA-YM?fur strain at 24 h and 48 h correspondingly.HenceRA-YM?fur strain decline virulence compared to RA-YM strain.It is indicateded that Fur is a virulence factor of R.anatipestifer.2.Investigation of relgulation genes of FurWe have identified the iron-and Fur-regulated genes in RA by RNA-seq analysis using RA-YM?fur and RA-YM in iron-restricted and iron-rich condition.The expression of 8 genes randomly selected which regulated by iron and Fur was confirmed by real-time PCR in RA-YM and RA-YM?fur in iron-restricted and iron-rich condition.The real-time PCR result was in accordance to the transcriptional data.In total,70 genes regulated by iron and 17 genes was regulated by Fur was screened out.And the specfic result of RNA-seq was shown as follows:The resulted showd there were 45 genes upregulated when grown in iron-restricted condition in both RA-YM and RA-YM?fur.Among the 45 upregualted genes,seven genes were involved in iron transport and acquire,five genes invovled in biological nitrogen fixation process,five genes participated in cell envelope and surface structure formation,2 genes participated in purine and nucleotide biosynthesis,others participated in macromolecules protein synthesis,transport and binding proteins and unknown function.And there were 25 genes downregulated when grown in iron-restricted condition in both RA-YM and RA-YM?fur.Among the 25 downregulated genes,six genes involved in tricarboxylic acid cycle,7 genes involved in respiration and oxidative stress,others involved in cell transport and unknown function.In addition,there were 17 genes directly regulated by Fur.Among the 17 genes regulated by Fur,five genes involved in iron acquirement,two genes involved in oxidative stress,one gene related to IX secretion system,others involved in amino acid synthesis and unknown function.The promoter regions of genes were analysed.And the predictive Fur box is 5'-ATTTAGAATTATTATAAAT-3'.The prokaryotic expressing of Fur protein was performed using plasmid p ET-28 a.The coding region of fur gene was amplified and then subcloned into plasmid p ET-28 a.The prokaryotic expression vector p ET28a-fur was transformed into competent cells of E.coil BL21(DE3).Then the Fur protein was induced by IPTG and purified with GE His Trap.Moreover,the promoter of genes were randomly selected which regulated by Fur was amplified using biotin labeled primers.The interaction between the Fur protein and the promoter of the genes regulated by Fur were verified by EMSA.The result showed that the promoter of the genes which regulated by Fur can bind to the purified Fur protein.In summary,we constructed an unmarkd gene deletion system containing counter-selectable marker phe S and indicated marker lac Z.And we constructed fur gene deletion mutant strain RA-YM?fur.And the complemented strain C?fur was constructed using shuttle plasmid p RES-JX-spc.It was also verified that fur gene was a virulence factor of RA by animal experiment.We have identified the iron-and Fur-regulated genes in RA by RNA-seq analysis using RA-YM?fur and RA-YM in iron-restricted and iron-rich condition.We also verified the putative Fur-box sequence was5'-ATTTAGAATTATTATAAAT-3'.Fur affects the virulence of RA by following ways:Fur regulated the iron acquirement system,oxidation-reduction reaction and IX secretion system,and then has influence on tricarboxylic acid cycle,purine and nucleotide biosynthesis.