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.
引文
[1]Nelson J J,Nelson C A,Carter J E.Extraintestinal manifestations of Edwardsiella tarda infection:A 10-year retrospective review[J].J La State Med Soc,2009,161(2):103-106.
[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.