冷冻致亚致死损伤的金黄色葡萄球菌修复机制
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摘要
【目的】研究冷冻致亚致死损伤的金黄色葡萄球菌修复过程中的细胞修复机制。【方法】本文以冷冻致亚致死损伤的金黄色葡萄球菌(Staphylococcus aureus)为研究对象,探讨了不同修复时间细胞的修复情况;利用透射电子显微镜观察修复启动过程中超微结构的变化;通过实时荧光定量PCR(Real-time PCR)方法测定了修复过程中转录弱化子(msrR)、铁离子ABC转运ATP结合蛋白(fhu C)、细胞色素b(cyt B)基因表达量的变化,通过紫外分光光度法测定细胞外泄漏物含量、细胞活性氧(ROS)和超氧化物歧化酶(SOD)活性。【结果】修复3 h后,99%以上冷冻致亚致死损伤的金黄色葡萄球菌完成修复,修复后细胞对高盐胁迫抗性恢复。Real-time PCR分析结果表明,msrR和fhu C基因表达量显著下调,而cyt B表达量显著上调。修复过程中冷冻致亚致死损伤的金黄色葡萄球菌细胞表面超微结构变化比较明显,细胞表面从光滑透明变得致密结实,细胞内紫外吸收物质泄漏速度也在逐渐变慢,同时细胞中的ROS含量降低,SOD酶活性减弱。【结论】冷冻致亚致死损伤的金黄色葡萄球菌修复过程中,可能是通过细胞膜完整性的修复,细胞恢复对高盐胁迫的抵抗能力;通过基因调控降低细胞内ROS的含量,降低活性氧(O-2)对细胞的毒害作用。同时通过产能代谢相关基因(cyt B)的调控为细胞提供修复所需要的能量,最终冷冻致亚致死损伤的细胞得到修复。
[Objective] To study the repair mechanisms of frozen sublethally damaged Staphylococcus aurous cells.[Methods]We resuscitated frozen sublethally damaged S. aureus at 37 ℃ for different time within 3 h. Meanwhile,we compared the morphological changes of the frozen sublethally damaged cells after 1 h of resuscitation using transmission electron microscopy assay( TEM). The expressions of the transcriptional attenuator MsrR( msrR),iron( Fe3 +) ABC transporter ATP-binding protein( fhu C),and cytochrome b( cyt B) genes were quantitatively analyzed by real-time fluorescence quantitative PCR( Real-time PCR) method. The content of cells outside leakage,active oxygen( ROS),and superoxide dismutase( SOD) activity were also determined by ultraviolet spectrophotometry. [Results]More than 99% of the frozen sublethally damaged S. aureus repaired after 3 h. The resuscitated cells expressed an equal resistance to high concentration of Na Cl. Real-time PCR results showed that the msrR and fhu C genes expressions were down-regulated,whereas the cyt B gene expression was up-regulated significantly. The frozen sublethally damaged S. aureus cellar surface ultrastructure significant changed during resuscitation. The cell surface became compact and sturdy from smooth and transparent. The cell leakage rate of ultraviolet absorption material gradually decreased. Meanwhile,the intracellular ROS level declined along with the decrease of SOD activity. [Conclusion] Frozen sublethally damaged cells may regain the capability of resistance to high salt stress by repairing cell membrane integrity,reducing the content of ROS through gene regulation,inhibiting the toxicity of active oxygen to the cells. Meanwhile,the regulation of metabolism related genes( cyt B) provides the energy for the requirement of cells,therefore,the frozen sublethally damaged cells were repaired finally.
[Objective] To study the repair mechanisms of frozen sublethally damaged Staphylococcus aurous cells.[Methods]We resuscitated frozen sublethally damaged S. aureus at 37 ℃ for different time within 3 h. Meanwhile,we compared the morphological changes of the frozen sublethally damaged cells after 1 h of resuscitation using transmission electron microscopy assay( TEM). The expressions of the transcriptional attenuator MsrR( msrR),iron( Fe3 +) ABC transporter ATP-binding protein( fhu C),and cytochrome b( cyt B) genes were quantitatively analyzed by real-time fluorescence quantitative PCR( Real-time PCR) method. The content of cells outside leakage,active oxygen( ROS),and superoxide dismutase( SOD) activity were also determined by ultraviolet spectrophotometry. [Results]More than 99% of the frozen sublethally damaged S. aureus repaired after 3 h. The resuscitated cells expressed an equal resistance to high concentration of Na Cl. Real-time PCR results showed that the msrR and fhu C genes expressions were down-regulated,whereas the cyt B gene expression was up-regulated significantly. The frozen sublethally damaged S. aureus cellar surface ultrastructure significant changed during resuscitation. The cell surface became compact and sturdy from smooth and transparent. The cell leakage rate of ultraviolet absorption material gradually decreased. Meanwhile,the intracellular ROS level declined along with the decrease of SOD activity. [Conclusion] Frozen sublethally damaged cells may regain the capability of resistance to high salt stress by repairing cell membrane integrity,reducing the content of ROS through gene regulation,inhibiting the toxicity of active oxygen to the cells. Meanwhile,the regulation of metabolism related genes( cyt B) provides the energy for the requirement of cells,therefore,the frozen sublethally damaged cells were repaired finally.
引文
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[11]Zhao W,Yang RJ,Zhang HQ,Zhang WB,Hua X,Tang YL.Quantitative and real time detection of pulsed electric field induced damage on Escherichia coli cells and sublethally injured microbial cells using flow cytometry in combination with fluorescent techniques.Food Control,2011,22(3/4):566-573.
[12]Xie YP,He YP,Irwin PL,Jin T,Shi XM.Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni.Applied and Environmental Microbiology,2011,77(7):2325-2331.
[13]Hübscher J,Mc Callum N,Sifri CD,Majcherczyk PA,Entenza JM,Heusser R,Berger-Bchi B,Meier PS.MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.Federation of European Microbiological Societies,2009,295(2):251-260.
[14]Sebulsky MT,Hohnstein D,Hunter MD,Heinrichs DE.Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus.Journal of Bacteriology,2000,182(16):4394-4400.
[15]Zwiers PB,Borgia G,Fleischer RC.Plumage based classification of the bowerbird genus sericulus evaluated using a multi-gene,multi-genome analysis.Molecular Phylogenetics and Evolution,2008,46(3):923-931.
[16]Matsuda K,Tsuji H,Asahara T,Kado Y,Nomoto K.Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR.Applied and Environmental Microbiology,2007,73(1):32-39.
[17]Loewen PC,Hengge-Aronis R.The role of the sigma factor sigmas(Kat F)in bacterial global regulation.Annual Review of Microbiology,1994,48(1):53-80.
[18]Lu HX,Huang XM,Zhu JL.Effect of ultra high pressure on cell membrane destruction and death of Listeria monocytogenes.Acta Microbiologica Sinica,2014,54(7):746-753.(in Chinese)陆海霞,黄小鸣,朱军莉.超高压对单增李斯特菌细胞膜的损伤和致死机理.微生物学报,2014,54(7):746-753.
[19]Becerra MC,Albesa I.Oxidative stress induced by ciprofloxacin in Staphylococcus aureus.Biochemical and Biophysical Research Communications,2002,297(4):1003-1007.
[20]Krishna MC,Russo A,Mitchell JB,Goldstein S,Dafni H,Samuni A.Do nitroxide antioxidants act as scavengers of O2-or as SOD mimics?The Journal of Biological Chemistry,1996,271(42):26026-26031.
[21]Vir P,Gupta D,Agarwal R,Verma I.Immunomodulation of alveolar epithelial cells by Mycobacterium tuberculosis phosphatidylinositol mannosides results in apoptosis.APMIS,2014,122(4):268-282.
[22]Rotruck JT,Pope AL,Ganther HE,Swanson AB,Hafeman DG,Hoekstra WG.Selenium:biochemical role as a component of glutathione peroxidase.Science,1973,179(4073):588-590.
[23]Over B,Heusser R,Mc Callum N,Schulthess B,Kupferschmied P,Gaiani JM,Sifri CD,Berger-Bchi B,Meier PS.LytR-Cps A-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation.FEMS Microbiology Letters,2011,320(2):142-151.
[24]Dengler V,Meier PS,Heusser R,Kupferschmied P,Fazekas J,Friebe S,Staufer SB,Majcherczyk PA,Moreillon P,Berger-Bchi B,Mc Callum N.Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response.FEMS Microbiology Letters,2012,333(2):109-120.
[25]Rossi J,Bischoff M,Wada A,Berger-Bchi B.MsrR,a putative cell envelope-associated element involved in Staphylococcus aureus sar A attenuation.Antimicrobial Agents and Chemotherapy,2003,47(8):2558-2564.
[26]Agranoff DD,Krishna S.Metal ion homeostasis and intracellular parasitism.Molecular Microbiology,1998,28(3):403-412.
[27]Cabrera G,Xiong AM,Uebel M,Singh VK,Jayaswal RK.Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus.Applied and Environmental Microbiology,2001,67(2):1001-1003.
[28]Burkhardt R,Braun V.Nucleotide sequence of the fhu C and fhu D genes involved in iron(III)hydroxamate transport:Domains in Fhu C homologous to ATP-binding proteins.Molecular and General Genetics,1987,209(1):49-55.
[29]Wallis MG,Groudinsky O,Slonimski PP,Dujiardin G.The NAM1 protein(NAM1p),which is selectively required for coxl,cytb and alp6 transcript processing/stabilisation,is located in the yeast mitochondrial matrix.European Journal of Biochemistry,1994,222(1):27-32.
[30]Taylor CT,Moncada S.Nitric oxide,cytochrome C oxidase,and the cellular response to hypoxia.Arteriosclerosis,Thrombosis,and Vascular Biology,2010,30(4):643-647.
[31]Mihoub M,Aloui A,El May A,Sethom MM,ben Ammar A,Jaafoura MH,Kacem R,Landoulsi A.Effects of garlic extract on cell wall of Corynebacterium glutamicum.Annual of Microbiology,2010,60(4):637-643.
[32]Carranza P,Grunau A,Schneider T,Hartmann I,Lehner A,Stephan R,Gehrig P,Grossmann J,Groebel K,Hoelzle LE,Eberl L,Riedel K.A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis 3032.Proteomics,2010,10(18):3248-3261.
[33]Zhou W,Liu GR,Li PL,Dai YQ,Zhou K.Mode of action of plantaricin L-1,an antilisteria bacteriocin produced by Lactobacillus plantarum.Acta Microbiologica Sinica,2007,47(2):260-264.(in Chinese)周伟,刘国荣,李平兰,戴蕴青,周康.植物乳杆菌素L-1对单核细胞增生李斯特氏菌作用机理的研究.微生物学报,2007,47(2):260-264.
[2]Schmitt M,Schuler-Schmid U,Schmidt-Lorenz W.Temperature limits of growth,TNase and enterotoxin production of Staphylococcus aureus strains isolated from foods.International Journal of Food Microbiology,1990,11(1):1-19.
[3]van Schothorst M,Zwietering MH,Ross T,Buchanan RL,Cole MB.International commission on microbiological specifications for foods(ICMSF).Relating microbiological criteria to food safety objectives and performance objectives.Food Control,2009,20(11):967-979.
[4]Suo B,Wang XJ,Pan ZL,Wang N,Ai ZL,Yu SJ,Salazar JK.Inactivation and sublethal injury kinetics of Staphylococcus aureus in broth at low temperature storage.Journal of Food Protection,2014,77(10):1689-1695.
[5]Sale AJH,Hamilton WA.Effects of high electric fields on microorganisms:I.Killing of bacteria and yeasts.Biochimica et Biophysica Acta(BBA)-General Subjects,1967,148(3):781-788.
[6]Wesche AM,Gurtler JB,Marks BP,Ryser ET.Stress,sublethal injury,resuscitation,and virulence of bacterial foodborne pathogens.Journal of Food Protection,2009,72(5):1121-1138.
[7]Mastronicolis SK,Diakogiannis I,Berberi A,Bisbiroulas P,Soukoulis C,Tzia C.Effect of cold adaptation on the survival of Listeria monocytogenes in ice-cream formulations during long-term frozen storage.Annals of Microbiology,2011,61(4):931-937.
[8]Brashears MM,Amezquita A,Stratton J.Validation of methods used to recover Escherichia coli O157:H7 and Salmonella spp.subjected to stress conditions.Journal of Food Protection,2001,64(10):1466-1471.
[9]Noriega E,Velliou E,Van Derlinden E,Mertens L,Van Impe JFM.Effect of cell immobilization on heat-induced sublethal injury of Escherichia coli,Salmonella typhimurium and Listeria innocua.Food Microbiology,2013,36(2):355-364.
[10]Suo B,Shi CL,Shi XM.Inactivation and occurrence of sublethal injury of Salmonella Typhimurium under mild heat stress in broth.Journal für Verbraucherschutz und Lebensmittelsicherheit,2012,7(2):125-131.
[11]Zhao W,Yang RJ,Zhang HQ,Zhang WB,Hua X,Tang YL.Quantitative and real time detection of pulsed electric field induced damage on Escherichia coli cells and sublethally injured microbial cells using flow cytometry in combination with fluorescent techniques.Food Control,2011,22(3/4):566-573.
[12]Xie YP,He YP,Irwin PL,Jin T,Shi XM.Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni.Applied and Environmental Microbiology,2011,77(7):2325-2331.
[13]Hübscher J,Mc Callum N,Sifri CD,Majcherczyk PA,Entenza JM,Heusser R,Berger-Bchi B,Meier PS.MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.Federation of European Microbiological Societies,2009,295(2):251-260.
[14]Sebulsky MT,Hohnstein D,Hunter MD,Heinrichs DE.Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus.Journal of Bacteriology,2000,182(16):4394-4400.
[15]Zwiers PB,Borgia G,Fleischer RC.Plumage based classification of the bowerbird genus sericulus evaluated using a multi-gene,multi-genome analysis.Molecular Phylogenetics and Evolution,2008,46(3):923-931.
[16]Matsuda K,Tsuji H,Asahara T,Kado Y,Nomoto K.Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR.Applied and Environmental Microbiology,2007,73(1):32-39.
[17]Loewen PC,Hengge-Aronis R.The role of the sigma factor sigmas(Kat F)in bacterial global regulation.Annual Review of Microbiology,1994,48(1):53-80.
[18]Lu HX,Huang XM,Zhu JL.Effect of ultra high pressure on cell membrane destruction and death of Listeria monocytogenes.Acta Microbiologica Sinica,2014,54(7):746-753.(in Chinese)陆海霞,黄小鸣,朱军莉.超高压对单增李斯特菌细胞膜的损伤和致死机理.微生物学报,2014,54(7):746-753.
[19]Becerra MC,Albesa I.Oxidative stress induced by ciprofloxacin in Staphylococcus aureus.Biochemical and Biophysical Research Communications,2002,297(4):1003-1007.
[20]Krishna MC,Russo A,Mitchell JB,Goldstein S,Dafni H,Samuni A.Do nitroxide antioxidants act as scavengers of O2-or as SOD mimics?The Journal of Biological Chemistry,1996,271(42):26026-26031.
[21]Vir P,Gupta D,Agarwal R,Verma I.Immunomodulation of alveolar epithelial cells by Mycobacterium tuberculosis phosphatidylinositol mannosides results in apoptosis.APMIS,2014,122(4):268-282.
[22]Rotruck JT,Pope AL,Ganther HE,Swanson AB,Hafeman DG,Hoekstra WG.Selenium:biochemical role as a component of glutathione peroxidase.Science,1973,179(4073):588-590.
[23]Over B,Heusser R,Mc Callum N,Schulthess B,Kupferschmied P,Gaiani JM,Sifri CD,Berger-Bchi B,Meier PS.LytR-Cps A-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation.FEMS Microbiology Letters,2011,320(2):142-151.
[24]Dengler V,Meier PS,Heusser R,Kupferschmied P,Fazekas J,Friebe S,Staufer SB,Majcherczyk PA,Moreillon P,Berger-Bchi B,Mc Callum N.Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response.FEMS Microbiology Letters,2012,333(2):109-120.
[25]Rossi J,Bischoff M,Wada A,Berger-Bchi B.MsrR,a putative cell envelope-associated element involved in Staphylococcus aureus sar A attenuation.Antimicrobial Agents and Chemotherapy,2003,47(8):2558-2564.
[26]Agranoff DD,Krishna S.Metal ion homeostasis and intracellular parasitism.Molecular Microbiology,1998,28(3):403-412.
[27]Cabrera G,Xiong AM,Uebel M,Singh VK,Jayaswal RK.Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus.Applied and Environmental Microbiology,2001,67(2):1001-1003.
[28]Burkhardt R,Braun V.Nucleotide sequence of the fhu C and fhu D genes involved in iron(III)hydroxamate transport:Domains in Fhu C homologous to ATP-binding proteins.Molecular and General Genetics,1987,209(1):49-55.
[29]Wallis MG,Groudinsky O,Slonimski PP,Dujiardin G.The NAM1 protein(NAM1p),which is selectively required for coxl,cytb and alp6 transcript processing/stabilisation,is located in the yeast mitochondrial matrix.European Journal of Biochemistry,1994,222(1):27-32.
[30]Taylor CT,Moncada S.Nitric oxide,cytochrome C oxidase,and the cellular response to hypoxia.Arteriosclerosis,Thrombosis,and Vascular Biology,2010,30(4):643-647.
[31]Mihoub M,Aloui A,El May A,Sethom MM,ben Ammar A,Jaafoura MH,Kacem R,Landoulsi A.Effects of garlic extract on cell wall of Corynebacterium glutamicum.Annual of Microbiology,2010,60(4):637-643.
[32]Carranza P,Grunau A,Schneider T,Hartmann I,Lehner A,Stephan R,Gehrig P,Grossmann J,Groebel K,Hoelzle LE,Eberl L,Riedel K.A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis 3032.Proteomics,2010,10(18):3248-3261.
[33]Zhou W,Liu GR,Li PL,Dai YQ,Zhou K.Mode of action of plantaricin L-1,an antilisteria bacteriocin produced by Lactobacillus plantarum.Acta Microbiologica Sinica,2007,47(2):260-264.(in Chinese)周伟,刘国荣,李平兰,戴蕴青,周康.植物乳杆菌素L-1对单核细胞增生李斯特氏菌作用机理的研究.微生物学报,2007,47(2):260-264.