貉肠炎沙门菌htra基因缺失株的构建及生物学特性
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摘要
为研究HtrA蛋白对貉源肠炎沙门菌毒力和生物学特性的影响,本试验利用Red同源重组方法对HtrA蛋白编码基因htra进行敲除,并从生长特性、生化特性、耐pH应激与氧化应激、抗血清杀伤和菌株毒力等方面对突变菌株进行评价,结果显示:与野生型菌株相比,htra基因缺失株生长特性和生化特性无明显差异,但其对酸性和碱性环境的敏感性增加,对血清的耐受力下降,动物实验结果显示htra基因缺失后肠炎沙门菌对小鼠半数致死量有显著提高。本试验成功构建貉肠炎沙门菌htra基因缺失株,为研究HtrA蛋白的作用机制奠定了基础。
It was well established that HtrA plays critical role in bactreial growth and survive under various stress conditions,and knockout of htra gene leads to sharply attenuated virulence in Escherichia coli.To investigate the roles of HtrA protein fromSalmonella enteritidis,isolated from raccoon,in biological characteristics and virulence.The htra mutant of Salmonella enteritidis was constructed byλ-Red homologous recombination,and comparison of growth characteristics,biochemical characteristics,anti-stress of pH,survival in serum and bacterial virulence were performed between wild type strain and htra mutant.The data demonstrated that,the mutant showed no difference in growth characteristics and biochemical characteristics with wild type strains.But compared to the wild type strains,the mutant displayed an increased sensitivity to pH stress and serum.And the htra mutant showed an increased LD50,indicating the deficient virulence of mutant.In this study,we successfully construct the htra gene-deleted mutant of raccoon Salmonella enteritidis,providing foundation for exploring underlying mechanism of HtrA protein.
It was well established that HtrA plays critical role in bactreial growth and survive under various stress conditions,and knockout of htra gene leads to sharply attenuated virulence in Escherichia coli.To investigate the roles of HtrA protein fromSalmonella enteritidis,isolated from raccoon,in biological characteristics and virulence.The htra mutant of Salmonella enteritidis was constructed byλ-Red homologous recombination,and comparison of growth characteristics,biochemical characteristics,anti-stress of pH,survival in serum and bacterial virulence were performed between wild type strain and htra mutant.The data demonstrated that,the mutant showed no difference in growth characteristics and biochemical characteristics with wild type strains.But compared to the wild type strains,the mutant displayed an increased sensitivity to pH stress and serum.And the htra mutant showed an increased LD50,indicating the deficient virulence of mutant.In this study,we successfully construct the htra gene-deleted mutant of raccoon Salmonella enteritidis,providing foundation for exploring underlying mechanism of HtrA protein.
引文
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[7]张志强,吴同垒,李永慧,等.狐肺炎大肠杆菌HtrA蛋白原核表达及多抗血清制备[J].中国兽医学报,2017,37(7):1255-1260.
[8]姚丰华,董立伟,张钰,等.肠炎沙门菌hcp基因对生物被膜形成的影响分析[J].中国预防兽医学报,2014,34(7):534-537.
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[12]黎敏英,邓树轩.肠炎沙门菌感染与宿主的关系[J].畜牧与饲料科学,2009(6):61-62.
[13] DIETZ H H,CHRIEL M,ANDERSEN T H,et al.Outbreak of Salmonella Dublin-associated abortion in Danish fur farms[J].Can Vet J,2006,47(12):1201-1205.
[14] GORHAM J R,CORDY D R,QUORTRUP E R.Salmonella infections in mink and ferrets[J].Am J Vet Res,1949,10(35):183-192.
[15]王贝贝,吴淑燕,黄瑞.巨噬细胞在沙门菌感染中作用的研究新进展[J].微生物与感染,2012(4):239-242.
[16] ROY F,VANTERPOOL E,FLETCHER H M.HtrA in Porphyromonas gingivalis can regulate growth and gingipain activity under stressful environmental conditions[J].Microbiology,2006,152(Pt 11):3391-3398.
[17] SKORKO-GLONEK J,SOBIECKA-SZKATULA A,NARKIEWICZ J,et al.The proteolytic activity of the HtrA(DegP)protein from Escherichia coli at low temperatures[J].Microbiology,2008,154(Pt12):3649-3658.
[18] BOEHM M,LIND J,BACKERT S,et al.Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance,oxygen resistance,host cell adhesion,invasion,and transmigration[J].Eur J Microbiol Immunol(Bp),2015,5(1):68-80.
[2]杨元斌,宋启发,闫鹏,等.2003—2014年浙江省宁波市肠炎沙门菌分子分型及耐药特征研究[J].疾病监测,2016(12):1007-1011.
[3]郭梦尧,侯萍,王国卿.貉沙门菌病的诊断与治疗[J].黑龙江畜牧兽医,2007(10):82-83.
[4]王胜启,李爱华,秦东春.沙门菌感染机制及其检测技术的研究进展[J].口岸卫生控制,2014(5):47-54.
[5]CHANG Z.The function of the DegP(HtrA)protein:Protease versus chaperone[J].IUBMB Life,2016,68(11):904-907.
[6]YE M,SHARMA K,THAKUR M,et al.HtrA,a temperature-and stationary phase-activated protease involved in maturation of a key microbial virulence determinant,facilitates borrelia burgdorferi infection in mammalian Hosts[J].Infect Immun,2016,84(8):2372-2381.
[7]张志强,吴同垒,李永慧,等.狐肺炎大肠杆菌HtrA蛋白原核表达及多抗血清制备[J].中国兽医学报,2017,37(7):1255-1260.
[8]姚丰华,董立伟,张钰,等.肠炎沙门菌hcp基因对生物被膜形成的影响分析[J].中国预防兽医学报,2014,34(7):534-537.
[9]王晓春,焦扬,程瞾,等.肠炎沙门菌lpfC基因缺失株的构建及基本生物学特性研究[J].生物技术,2015(3):256-261.
[10] CHI F,WANG Y,GALLAHER T K,et al.Identification of IbeR as a stationary-phase regulator in meningitic Escherichia coli K1that carries a loss-of-function mutation in rpoS[J].J Biomed Biotechnol,2009,2009:520283.
[11] WANG C Y,WANG S W,HUANG W C,et al.Prc contributes to Escherichia coli evasion of classical complement-mediated serum killing[J].Infect Immun,2012,80(10):3399-3409.
[12]黎敏英,邓树轩.肠炎沙门菌感染与宿主的关系[J].畜牧与饲料科学,2009(6):61-62.
[13] DIETZ H H,CHRIEL M,ANDERSEN T H,et al.Outbreak of Salmonella Dublin-associated abortion in Danish fur farms[J].Can Vet J,2006,47(12):1201-1205.
[14] GORHAM J R,CORDY D R,QUORTRUP E R.Salmonella infections in mink and ferrets[J].Am J Vet Res,1949,10(35):183-192.
[15]王贝贝,吴淑燕,黄瑞.巨噬细胞在沙门菌感染中作用的研究新进展[J].微生物与感染,2012(4):239-242.
[16] ROY F,VANTERPOOL E,FLETCHER H M.HtrA in Porphyromonas gingivalis can regulate growth and gingipain activity under stressful environmental conditions[J].Microbiology,2006,152(Pt 11):3391-3398.
[17] SKORKO-GLONEK J,SOBIECKA-SZKATULA A,NARKIEWICZ J,et al.The proteolytic activity of the HtrA(DegP)protein from Escherichia coli at low temperatures[J].Microbiology,2008,154(Pt12):3649-3658.
[18] BOEHM M,LIND J,BACKERT S,et al.Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance,oxygen resistance,host cell adhesion,invasion,and transmigration[J].Eur J Microbiol Immunol(Bp),2015,5(1):68-80.