类鼻疽伯克霍尔德菌bopA基因敲除株的构建及生物学特征
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摘要
【【背景】类鼻疽杆菌是一种能够引起人类疾病甚至死亡的胞内寄生菌,Ⅲ型分泌系统在该菌入侵上皮细胞、逃避宿主免疫以及毒力因子的分泌过程中发挥重要作用,其中bopA基因为TTSS-3基因编码的重要效应蛋白,在类鼻疽杆菌的免疫逃逸中发挥重要作用。【目的】构建类鼻疽杆菌bopA基因敲除菌株,并对其生物学特征进行初步研究。【方法】构建pK18mobSacB-ΔbopA自杀质粒,通过大肠杆菌S17-1λpair以接合的方式转入类鼻疽杆菌,利用同源重组敲除了bopA基因,并用蔗糖平板筛选出菌株,最后在细胞和动物水平检测敲除菌株的表型变化。【结果】构建了bopA敲除的类鼻疽菌株,并通过细胞和动物实验证实敲除bopA基因后,细菌的细胞侵袭和胞内存活以及体内定殖能力都显著降低。【结论】利用同源重组成功构建类鼻疽bopA基因敲除株,为深入研究该基因的作用靶点奠定了实验基础。
[Background] Burkholderia pseudomallei is an intracellular parasitic bacterium that can causehuman disease and even death. The type III secretory system plays an important role in the bacterialinvasion of epithelial cells, escaping host immunity and the secretion of virulence factors. The bopA geneis an important effector protein encoded by the TTSS-3 gene, and it plays an important role in the immuneescape of the Burkholderia pseudomallei. [Objective] To construct Burkholderia pseudomallei bopA geneknockout mutant strain, and evaluate the biological characteristics of the mutant strain. [Methods] Weconstructed suicide plasmid pK18 mobSacB-ΔbopA, and then transformed the plasmid into B. pseudomalleifrom Escherichia coli S17-1λpair by conjugation. bopA gene was knocked out by homologousrecombination, and the mutant strain was selected by sucrose agar screening. The phenotypic variation ofthe mutant strain was finally evaluated at the cell and animal levels. [Results] We constructed B.pseudomallei bopA mutant strain successfully, and found the invasion rate, intracellular survival ability aswell as the ability of colonization in vivo of mutant strain was significantly reduced. [Conclusion] Weconstructed B. pseudomallei bopA mutant strain by homologous recombination, to provide a basis forfurther understanding of this gene.
[Background] Burkholderia pseudomallei is an intracellular parasitic bacterium that can causehuman disease and even death. The type III secretory system plays an important role in the bacterialinvasion of epithelial cells, escaping host immunity and the secretion of virulence factors. The bopA geneis an important effector protein encoded by the TTSS-3 gene, and it plays an important role in the immuneescape of the Burkholderia pseudomallei. [Objective] To construct Burkholderia pseudomallei bopA geneknockout mutant strain, and evaluate the biological characteristics of the mutant strain. [Methods] Weconstructed suicide plasmid pK18 mobSacB-ΔbopA, and then transformed the plasmid into B. pseudomalleifrom Escherichia coli S17-1λpair by conjugation. bopA gene was knocked out by homologousrecombination, and the mutant strain was selected by sucrose agar screening. The phenotypic variation ofthe mutant strain was finally evaluated at the cell and animal levels. [Results] We constructed B.pseudomallei bopA mutant strain successfully, and found the invasion rate, intracellular survival ability aswell as the ability of colonization in vivo of mutant strain was significantly reduced. [Conclusion] Weconstructed B. pseudomallei bopA mutant strain by homologous recombination, to provide a basis forfurther understanding of this gene.
引文
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[10]Hu ZQ,Fang Y,Zhu P,et al.Construction and identification of Burkholderia pseudomallei BPSL1549gene knockout mutant strain[J].Journal of Third Military Medical University,2016,38(11):1210-1214(in Chinese)胡治强,方瑶,朱攀,等.类鼻疽伯克霍尔德菌BPSL1549基因敲除株的构建及鉴定[J].第三军医大学学报,2016,38(11):1210-1214
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[12]Kayath CA,Hussey S,El Hajjami N,et al.Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector,IcsB[J].Microbes and Infection,2010,12(12/13):956-966
[13]Yu D,Yin ZQ,Jin Y,et al.Evolution of bopA gene in Burkholderia:a case of convergent evolution as a mechanism for bacterial autophagy evasion[J].BioMed Research International,2016,2016:6745028
[2]Mao XH.Progress on melioidosis[J].Journal of Third Military Medical University,2011,33(13):1315-1317(in Chinese)毛旭虎.加强类鼻疽的研究[J].第三军医大学学报,2011,33(13):1315-1317
[3]Ren CY,Li Q.A lethal toxin from Burkholderia pseudomallei[J].Progress in Microbiology and Immunology,2015,43(3):47-50(in Chinese)任春艳,李倩.类鼻疽伯克霍尔德致死因子[J].微生物学免疫学进展,2015,43(3):47-50
[4]Ulrich RL,DeShazer D.Type III secretion:a virulence factor delivery system essential for the pathogenicity of Burkholderia mallei[J].Infection and Immunity,2004,72(2):1150-1154
[5]Galyov EE,Brett PJ,DeShazer D.Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis[J].Annual Review of Microbiology,2010,64:495-517
[6]Stevens MP,Haque A,Atkins T,et al.Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis[J].Microbiology,2004,150(8):2669-2676
[7]Cullinane M,Gong L,Li XL,et al.Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines[J].Autophagy,2008,4(6):744-753
[8]Sch?fer A,Tauch A,J?ger W,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J].Gene,1994,145(1):69-73
[9]Gong L,Cullinane M,Treerat P,et al.The Burkholderia pseudomallei type III secretion system and bopA are required for evasion of LC3-associated phagocytosis[J].PLoS One,2011,6(3):e17852
[10]Hu ZQ,Fang Y,Zhu P,et al.Construction and identification of Burkholderia pseudomallei BPSL1549gene knockout mutant strain[J].Journal of Third Military Medical University,2016,38(11):1210-1214(in Chinese)胡治强,方瑶,朱攀,等.类鼻疽伯克霍尔德菌BPSL1549基因敲除株的构建及鉴定[J].第三军医大学学报,2016,38(11):1210-1214
[11]Pei JM,Grishin NV.The Rho GTPase inactivation domain in Vibrio cholerae MARTX toxin has a circularly permuted papain-like thiol protease fold[J].Proteins,2009,77(2):413-419
[12]Kayath CA,Hussey S,El Hajjami N,et al.Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector,IcsB[J].Microbes and Infection,2010,12(12/13):956-966
[13]Yu D,Yin ZQ,Jin Y,et al.Evolution of bopA gene in Burkholderia:a case of convergent evolution as a mechanism for bacterial autophagy evasion[J].BioMed Research International,2016,2016:6745028