狐源大肠杆菌HtrA基因缺失株的构建及生物学特性
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摘要
HtrA是一种重要的抗应激蛋白,对细菌抵抗应激条件至关重要。为研究HtrA蛋白在狐源大肠杆菌致病过程中的作用,本研究利用λ-Red同源重组方法构建狐大肠杆菌HtrA基因缺失株,并从生长特性、生化特性、抗吞噬细胞吞噬能力、菌株毒力等几方面对突变菌株进行评价,结果表明:与狐源大肠杆菌野生型菌株相比,HtrA基因缺失株生长特性和生化特性无明显差异,但其抗吞噬细胞吞噬能力有明显下降,细菌半数致死量有显著提高。本研究成功构建狐源大肠杆菌HtrA基因缺失株,为研究HtrA蛋白的作用机制奠定了基础。
HtrA,an important stress response protein,plays critical role in growth and survive under various stress conditions.To investigate the roles of HtrA protein in pathogenic processes of E.coli,λ-Red homologous recombination was used for knockout of HtrA gene,and comparison of growth characteristics,biochemical characteristics,anti-phagocytosis ability and bacterial virulence were performed between E.coli wild type strain and HtrA knockout 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,anti-phagocytosis ability were sharply impired,and LD_(50) was significantly increased,indicating the deficient virulence of mutant.In this study,the HtrA gene-deleted mutant of E.coli were successfully constructed,providing foundation for exploring underlying mechanism of HtrA protein.
HtrA,an important stress response protein,plays critical role in growth and survive under various stress conditions.To investigate the roles of HtrA protein in pathogenic processes of E.coli,λ-Red homologous recombination was used for knockout of HtrA gene,and comparison of growth characteristics,biochemical characteristics,anti-phagocytosis ability and bacterial virulence were performed between E.coli wild type strain and HtrA knockout 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,anti-phagocytosis ability were sharply impired,and LD_(50) was significantly increased,indicating the deficient virulence of mutant.In this study,the HtrA gene-deleted mutant of E.coli were successfully constructed,providing foundation for exploring underlying mechanism of HtrA protein.
引文
[1] 曲海娜,邹玲,刘文华,等.毛皮动物细菌病流行病学调查及耐药分析[J].中国动物检疫,2009(2):48-50.
[2] 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.
[3] 于杰,曲海娜,张灿,等.毛皮动物大肠杆菌对氟喹诺酮类药物及中药的耐药性研究[J].中国畜牧兽医,2015(6):1597-1601.
[4] 钟世勋,迟珊珊,王振,等.山东规模化养殖场毛皮动物多重感染病原学分析[J].中国兽医学报,2014,34(11):1770-1777.
[5] PALLEN M J,WREN B W.The HtrA family of serine proteases[J].Mol Microbiol,1997,26(2):209-221.
[6] 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.
[7] BAEK K T,VEGGE C S,SKORKO-GLONEK J,et al.Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology[J].Appl Environ Microbiol,2011,77(1):57-66.
[8] CHI F,WANG Y,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].J Biomed Biotechnol,2009,2009:520283.
[9] ZURAWA-JANICKA D,NARKIEWICZ J,LIPINSKA B.Characterization of the HtrA family of proteins[J].Postepy Biochem,2007,53(1):27-36.
[10] 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.
[11] 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(Pt 12):3649-3658.
[12] KANG K H,LEE J S,YOO M,et al.The influence of HtrA expression on the growth of Streptococcus mutans during acid stress[J].Mol Cells,2010,29(3):297-304.
[13] FLANNAGAN R S,AUBERT D,KOOI C,et al.Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo[J].Infect Immun,2007,75(4):1679-1689.
[14] SKORKO-GLONEK J,ZURAWA D,KUCZWARA E,et al.The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress[J].Mol Gen Genet,1999,262(2):342-350.
[15] BRINGER M A,BARNICH N,GLASSER A L,et al.HtrA stress protein is involved in intramacrophagic replication of adherent and invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease[J].Infect Immun,2005,73(2):712-721.
[16] DE STOPPELAAR S F,BOOTSMA H J,ZOMER A,et al.Streptococcus pneumoniae serine protease HtrA,but not SFP or PrtA,is a major virulence factor in pneumonia[J].PLoS One,2013,8(11):e80062.
[17] CHITLARU T,ZAIDE G,EHRLICH S,et al.HtrA is a major virulence determinant of Bacillus anthracis[J].Mol Microbiol,2011,81(6):1542-1559.
[18] WILSON R L,BROWN L L,KIRKWOOD-WATTS D,et al.Listeria monocytogenes 10403S HtrA is necessary for resistance to cellular stress and virulence[J].Infect Immun,2006,74(1):765-768.
[19] LEWIS C,SKOVIEROVA H,ROWLEY G,et al.Salmonella enterica Serovar Typhimurium HtrA:regulation of expression and role of the chaperone and protease activities during infection[J].Microbiology,2009,155(Pt 3):873-881.
[20] ZHU C,XIONG K,CHEN Z,et al.Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD[J].Microbiol Immunol,2015,59(8):443-451.
[2] 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.
[3] 于杰,曲海娜,张灿,等.毛皮动物大肠杆菌对氟喹诺酮类药物及中药的耐药性研究[J].中国畜牧兽医,2015(6):1597-1601.
[4] 钟世勋,迟珊珊,王振,等.山东规模化养殖场毛皮动物多重感染病原学分析[J].中国兽医学报,2014,34(11):1770-1777.
[5] PALLEN M J,WREN B W.The HtrA family of serine proteases[J].Mol Microbiol,1997,26(2):209-221.
[6] 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.
[7] BAEK K T,VEGGE C S,SKORKO-GLONEK J,et al.Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology[J].Appl Environ Microbiol,2011,77(1):57-66.
[8] CHI F,WANG Y,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].J Biomed Biotechnol,2009,2009:520283.
[9] ZURAWA-JANICKA D,NARKIEWICZ J,LIPINSKA B.Characterization of the HtrA family of proteins[J].Postepy Biochem,2007,53(1):27-36.
[10] 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.
[11] 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(Pt 12):3649-3658.
[12] KANG K H,LEE J S,YOO M,et al.The influence of HtrA expression on the growth of Streptococcus mutans during acid stress[J].Mol Cells,2010,29(3):297-304.
[13] FLANNAGAN R S,AUBERT D,KOOI C,et al.Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo[J].Infect Immun,2007,75(4):1679-1689.
[14] SKORKO-GLONEK J,ZURAWA D,KUCZWARA E,et al.The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress[J].Mol Gen Genet,1999,262(2):342-350.
[15] BRINGER M A,BARNICH N,GLASSER A L,et al.HtrA stress protein is involved in intramacrophagic replication of adherent and invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease[J].Infect Immun,2005,73(2):712-721.
[16] DE STOPPELAAR S F,BOOTSMA H J,ZOMER A,et al.Streptococcus pneumoniae serine protease HtrA,but not SFP or PrtA,is a major virulence factor in pneumonia[J].PLoS One,2013,8(11):e80062.
[17] CHITLARU T,ZAIDE G,EHRLICH S,et al.HtrA is a major virulence determinant of Bacillus anthracis[J].Mol Microbiol,2011,81(6):1542-1559.
[18] WILSON R L,BROWN L L,KIRKWOOD-WATTS D,et al.Listeria monocytogenes 10403S HtrA is necessary for resistance to cellular stress and virulence[J].Infect Immun,2006,74(1):765-768.
[19] LEWIS C,SKOVIEROVA H,ROWLEY G,et al.Salmonella enterica Serovar Typhimurium HtrA:regulation of expression and role of the chaperone and protease activities during infection[J].Microbiology,2009,155(Pt 3):873-881.
[20] ZHU C,XIONG K,CHEN Z,et al.Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD[J].Microbiol Immunol,2015,59(8):443-451.