迟缓爱德华菌menF基因缺失株的构建及生物学特性研究
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摘要
为研究异分支酸合成酶MenF在迟缓爱德华菌(E.tarda)致病中的作用,本研究利用自杀载体构建了E.tarda menF基因缺失菌株(ET-CLΔmenF),并分析其生物学特性。结果显示,ET-CLΔmenF株比野生菌株生长减缓。体外应激试验结果表明,ET-CLΔmenF菌株对铁饥饿的耐受能力明显下降,应对酸性和过氧化氢处理的能力显著下降。细胞感染实验表明ET-CLΔmenF菌株的胞内存活能力显著下降(p<0.01)。对斑马鱼的致病性结果表明,ET-CLΔmenF株毒力下降。本研究为E.tarda致病机制研究以及疫苗的研制提供了参考。
To investigate the role of isochorismate synthase MenF in Edwardsiella tarda, we generated a MenF-knockout mutant with the suicide plasmid pRE112 and characterized the biochemical properties of the mutant. The data showed that, the MenF-knockout strain led to retarded growth in LB and Minimal(M9) medium. Furthermore, the MenF-deficient mutant displayed sharply decreased survival under acidic and H_2O_2 treatments. The resistance to iron starvation was compromised in the MenF-deficient mutant, compared with the wild type strains. Moreover, MenF-inactivation-caused reduced cellular survival of E.tarda in Raw264.7 cells was determined. In addition, we found that MenF-knockout strain resulted in virulence attenuation in the zebra fish model. In conclusion, this study provided insight into the underlying mechanism of E.tarda infection in aquaculture.
To investigate the role of isochorismate synthase MenF in Edwardsiella tarda, we generated a MenF-knockout mutant with the suicide plasmid pRE112 and characterized the biochemical properties of the mutant. The data showed that, the MenF-knockout strain led to retarded growth in LB and Minimal(M9) medium. Furthermore, the MenF-deficient mutant displayed sharply decreased survival under acidic and H_2O_2 treatments. The resistance to iron starvation was compromised in the MenF-deficient mutant, compared with the wild type strains. Moreover, MenF-inactivation-caused reduced cellular survival of E.tarda in Raw264.7 cells was determined. In addition, we found that MenF-knockout strain resulted in virulence attenuation in the zebra fish model. In conclusion, this study provided insight into the underlying mechanism of E.tarda infection in aquaculture.
引文
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[11]Domagalski M J,Tkaczuk K L,Chruszcz M,et al.Structure of isochorismate synthase DhbC from Bacillus anthracis[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2013,69(Pt 9):956-961.
[12]Sridharan S,Howard N,Kerbarh O,et al.Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase(EntC)bound to its reaction product isochorismate:Implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes[J].J Mol Biol,2010,397(1):290-300.
[13]Pelludat C,Brem D,Heesemann J.Irp9,encoded by the high-pathogenicity island of Yersinia enterocolitica,is able to convert chorismate into salicylate,the precursor of the siderophore yersiniabactin[J].J Bacteriol,2003,185(18):5648-5653.
[14]Leung K Y,Siame B A,Tenkink B J,et al.Edwardsiella tarda-virulence mechanisms of an emerging gastroenteritis pathogen[J].Microbes Infect,2012,14(1):26-34.
[15]Mo Zhao-lan,Li Jie,Li Gui-yang,et al.Phenotypic characterization,virulence,and immunogenicity of Edwardsiella tarda LSE40 aroA mutant[J].Appl Microbiol Biotechnol,2013,97(14):6325-6335.
[2]Hirai Y,Asahata-Tago S,Ainoda Y,et al.Edwardsiella tarda bacteremia.A rare but fatal water-and foodborne infection:Review of the literature and clinical cases from a single centre[J].Can J Infect Dis Med Microbiol,2015,26(6):313-318.
[3]李杰.迟缓爱德华氏菌弱毒疫苗的研制和免疫效果[D].青岛:中国科学院研究生院(海洋研究所),2012.
[4]Han Xian-gan,Bai Hao,Tu Jian,et al.Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coliaroA mutant[J].Microb Pathog,2015,88:39-47.
[5]Matsui H,Isshiki Y,Eguchi M,et al.Evaluation of the live vaccine efficacy of virulence plasmid-cured,and phoP-or aroA-deficient Salmonella enterica serovar Typhimurium in mice[J].J Vet Med Sci,2015,77(2):181-186.
[6]Yao Zu-jie,Wang Zhi-hong,Sun Lin-an,et al.Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila[J].BMC Microbiol,2016,16(1):161.
[7]Chi Feng,Wang Ying,Gallaher T K,et al.Identification of IbeR as a stationary-phase regulator in meningitic Escherichia coli K1 that carries a loss-of-function mutation in rpoS[J].JBiomed Biotechnol,2009,2009:520283.
[8]Dong Hong-yan,Peng Da-xin,Jiao Xin-an,et al.Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence[J].Microbiology,2011,157(Pt 6):1798-1805.
[9]孙云.迟缓爱德华氏菌高效疫苗的设计、构建以及免疫机制研究[D].青岛:中国科学院研究生院(海洋研究所),2013.
[10]Buss K,Muller R,Dahm C,et al.Clustering of isochorismate synthase genes menF and entC and channeling of isochorismate in Escherichia coli[J].Biochim Biophys Acta,2001,1522(3):151-157.
[11]Domagalski M J,Tkaczuk K L,Chruszcz M,et al.Structure of isochorismate synthase DhbC from Bacillus anthracis[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2013,69(Pt 9):956-961.
[12]Sridharan S,Howard N,Kerbarh O,et al.Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase(EntC)bound to its reaction product isochorismate:Implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes[J].J Mol Biol,2010,397(1):290-300.
[13]Pelludat C,Brem D,Heesemann J.Irp9,encoded by the high-pathogenicity island of Yersinia enterocolitica,is able to convert chorismate into salicylate,the precursor of the siderophore yersiniabactin[J].J Bacteriol,2003,185(18):5648-5653.
[14]Leung K Y,Siame B A,Tenkink B J,et al.Edwardsiella tarda-virulence mechanisms of an emerging gastroenteritis pathogen[J].Microbes Infect,2012,14(1):26-34.
[15]Mo Zhao-lan,Li Jie,Li Gui-yang,et al.Phenotypic characterization,virulence,and immunogenicity of Edwardsiella tarda LSE40 aroA mutant[J].Appl Microbiol Biotechnol,2013,97(14):6325-6335.