羊种布鲁氏菌M5-90疫苗株铁应答调控因子irr和rirA基因缺失株的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2020.01.002

Abstract

Abstract: In order to explore the role of iron response regulator (irr) and rhizobial iron regulator (rirA) during Brucella melitensis infection,in this research,two mutants M5-90Δirr and M5-90ΔrirA were constructed through the kanamycin gene replacement method.These two mutants and their parental strain M5-90 were cultured for 36 h in the same condition,the growth ability were measured.1×10⁸ CFU M5-90Δirr,M5-90ΔrirA and M5-90 were inoculated into the 1 mL Brucella broth supplemented with 1.5 mmol/L NaCl,pH 2.5,pH 11.5 and 10 mmol/L H₂O₂ to detect the growth ability of these two mutants.The adhesion,invasion and intracellular survival ability of these two mutants were evaluated in RAW264.7 cells infected with 1×10⁶ CFU either M5-90Δirr,M5-90ΔrirA or M5-90.The results showed that under the same culture condition,the growth rates of two mutants M5-90Δirr and M5-90ΔrirA were significantly lower than that of their parental strain.The survival rates of M5-90Δirr and M5-90ΔrirA in high salt,strong acid and alkali environment were significantly lower than those in parental strains (P<0.05).The survival rates of these two mutants were significantly higher than the parental strain under hydrogen peroxide conditions (P<0.05).The invasion and adhesion capability of these two mutants inside the murine macrophages RAW264.7 were attenuated,especially at 45 min post infection,the invasion and adhesion capability of these two mutants were extremely significantly lower than M5-90 (P<0.01),however,24 h after infection,the ability of these two mutants to propagate in cells was enhanced compared with that of their parental strain.This was the first report that irr and rirA genes of B.melitensis not only associated with the virulence but also with the growth ability.To sum up,M5-90Δirr and M5-90ΔrirA were two promising Brucella vaccine candidates.

Key words: Brucella melitensis; iron response regulator; irr gene; rirA gene; virulence

CLC Number:

S852.61

ZHAO Xiaoli, WANG Benben, ZHANG Huan, YANG Yajun, YANG Ningning, WANG Zhen, YANG Qin, WANG Yong, CHEN Chuangfu, CAO Xudong. Construction and Identification of Iron Responsive Regulator irr and rirA Genes Deletion Strain of Brucella melitensis M5-90 Vaccine Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 8-17.

References

[1] LEON-SICAIROS N,ANGULO-ZAMUDIO U A,DE LA GARZA M,et al.Strategies of Vibrio parahaemolyticus to acquire nutritional iron during host colonization[J].Frontiers in Microbiology,2015,6:702.
[2] ANDREWS S C,ROBINSON A K.Bacterial iron homeostasis[J].FEMS Microbiology Reviews,2003,27(2-3):215-237.
[3] WALDRON K J,ROBINSON N J.How do bacterial cells ensure that metalloproteins get the correct metal?[J].Nature Reviews Microbiology,2009,7(1):25-35.
[4] ZHAO X,DRLICA K.Reactive oxygen species and the bacterial response to lethal stress[J].Current Opinion in Microbiology,2014,21:1-6.
[5] CHANDRANGSU P,RENSING C,HELMANN J D.Metal homeostasis and resistance in bacteria[J].Nature Reviews Microbiology,2017,15(6):338-350.
[6] PORCHERON G,DOZOIS C M.Interplay between iron homeostasis and virulence:Fur and RyhB as major regulators of bacterial pathogenicity[J].Veterinary Microbiology,2015,179(1-2):2-14.
[7] MARTINEZ M.Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis,and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus[J].Microbiology,2006,152(9):2591-2598.
[8] TODD J D,WEXLER M,SAWERS G,et al.RirA,an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum[J].Microbiology,2002,148(Pt 12):4059-4071.
[9] 王震,王勇,张倩,等.布鲁菌铁转录调控因子rirA基因突变株的构建与鉴定[J].动物医学进展,2017,38(10):1-7. WANG Z,WANG Y,ZHANG Q,et al.Construction and identification of Brucella rirA gene mutant[J].Progress in Veterinary Medicine,2017,38(10):1-7.(in Chinese)
[10] SCHAIBLE U E,KAUFMANN S H.Iron and microbial infection[J].Nature Reviews Microbiology,2004,2(12):946-953.
[11] GESINE R,HAUKE H,HANS-MARTIN F.Beyond the Fur paradigm:Iron-controlled gene expression in rhizobia[J].FEMS Microbiology Reviews,2010,30(4):631-648.
[12] MARCELA M,UGALDE R A,MARTA A.Dimeric Brucella abortus irr protein controls its own expression and binds haem[J].Microbiology,2005,151(Pt 10):3427-3433.
[13] ANDERSON E S,PAULLEY J T,MARTINSON D A,et al.The iron-responsive regulator irr is required for wild-type expression of the gene encoding the heme transporter BhuA in Brucella abortus 2308[J].Journal of Bacteriology,2011,193(19):5359-5364.
[14] LEE B C.Quelling the red menace:Haem capture by bacteria[J].Molecular Microbiology,2010,18(3):383-390.
[15] JOHNSTON A W B,TODD J D,CURSON A R,et al.Living without Fur:The subtlety and complexity of iron-responsive gene regulation in the symbiotic bacterium Rhizobium and other α-proteobacteria[J].Biometals,2007,20(3-4):501-511.
[16] RUDOLPH G,SEMINI G,HAUSER F,et al.The iron control element,acting in positive and negative control of iron-regulated Bradyrhizobium japonicum genes,is a target for the irr protein[J].Journal of Bacteriology,2006,188(2):733-744.
[17] RODIONOV D A,GELFAND M S,TODD J D,et al.Computational reconstruction of iron- and manganese-responsive transcriptional networks in α-proteobacteria[J].PLoS Computational Biology,2006,2(12):e163.
[18] TODD J D,SAWERS G,JOHNSTON A W.Proteomic analysis reveals the wide-ranging effects of the novel,iron-responsive regulator rirA in Rhizobium leguminosarum bv.viciae[J].Molecular Genetics Genomics,2005,273(2):197-206.
[19] ESCOLAR L,PÉREZ-MARTÍN J,DE LORENZO V.Opening the iron box:Transcriptional metalloregulation by the Fur protein[J].Journal of Bacteriology,1999,181(20):6223-6229.
[20] MAURICIO L,DANIELA Q,DANTE T,et al.The role of Fur in the transcriptional and iron homeostatic response of Enterococcus faecalis[J].Frontiers in Microbiology,2018,9:1580.
[21] PASQUA M,VISAGGI D,LO SCIUTO A,et al.Ferric uptake regulator Fur is conditionally essential in Pseudomonas aeruginosa[J].Journal of Bacteriology,2017,199(22):e00472-17.
[22] KURABAYASHI K,AGATA T,ASANO H,et al.Fur represses adhesion to,invasion of,and intracellular bacterial community formation within bladder epithelial cells and motility in uropathogenic Escherichia coli[J].Infection and Immunity,2016,84(11):3220-3231.
[23] RITCHIE J A,RUPPER A,CARDELLI J A,et al.Host interferon-γ inducible protein contributes to Brucella survival[J].Frontiers in Cellular and Infection Microbiology,2012,2:55.
[24] HIBBING M E,FUQUA C.Antiparallel and interlinked control of cellular iron levels by the irr and rirA regulators of Agrobacterium tumefaciens[J].Journal of Bacteriology,2011,193(14):3461-3472.

Construction and Identification of Iron Responsive Regulator irr and rirA Genes Deletion Strain of Brucella melitensis M5-90 Vaccine Strain

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