基于iTRAQ蛋白组学的植物乳杆菌镉吸附及耐受特征分析
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摘要
植物乳杆菌是在食品工业中广泛应用的一种益生菌,不同植物乳杆菌对重金属镉的吸附及耐受能力存在显著的菌株差异。为了探究植物乳杆菌的镉吸附机制,通过筛选得到两株镉吸附能力有显著差别的植物乳杆菌(强吸附菌株CCFM8610和弱吸附菌株CCFM595),采用基于同位素标记相对和绝对定量(iTRAQ)方法的比较蛋白组学分析手段,研究两株菌间的差异表达蛋白。在两菌株中共鉴定得到1 690个蛋白,其中有109个丰度差异表达蛋白,以及89个仅在强镉吸附菌株CCFM8610检测到的蛋白。通过对这些差异蛋白进行功能注释、代谢通路重构以及互作网络解析,阐明植物乳杆菌CCFM8610与CCFM595的镉吸附及耐受机制可能为:表达更多的参与细胞壁及胞外聚合物合成的蛋白,从而加强镉离子的隔离和吸附;CCFM8610一直处于低压力的状态,表现为压力应答相关蛋白在CCFM8610中低表达;CCFM8610具有独特的能量节省的代谢模式,表现为TCA循环通路蛋白的整体较低水平;CCFM8610具有更强的金属外排转运能力,表现为cadA等镉外排泵也在CCFM8610具有更高丰度;CCFM8610的疏水性氨基酸的表达增加,可增强细胞的表面疏水性,还能通过表达调节渗透压及胞内结合镉的氨基酸来应对镉毒性。
Lactobacillus plantarum is a probiotic bacteria widely used in food industry. There are significant differences in adsorption and tolerance of cadmium in different Lactobacillus plantarum strains. Two strains of Lactobacillus plantarum(strongly adsorbed strain CCFM8610 and weakly adsorbed strain CCFM595) with significant differences in cadmium adsorption capacity were screened. The differentially expressed proteins between the two strains were studied by comparative proteomic analysis based on isobaric tags for relative and absolute quantification(iTRAQ). The results showed that totally 1 690 proteins were identified in the two strains, 109 abundant differentially expressed proteins and89 proteins were exclusively detected in CCFM8610. Through functional annotation, metabolic pathway reconstruction and interaction network analysis of these differentially expressed proteins, the possible mechanisms of cadmium adsorption and tolerance of Lactobacillus plantarum CCFM8610 are as follows: it has a unique energy-saving metabolic pattern, which is characterized by the lower overall level of TCA cycle pathway proteins; expresses more proteins involved in cell wall and extracellular polymer synthesis, thus enhancing the isolation and adsorption of cadmium ions; it has stronger metal efflux transporting capacity, such as cadmium efflux pump such as cadmium also has higher abundance in CCFM8610;CCFM8610 shows higher expression of proteins involved in biosynthesis of hydrophobic amino acids, thus improving surface hydrophobicity of the strain and meanwhile upregulating certain amino acids to bind Cd to tackle Cd toxicity.
Lactobacillus plantarum is a probiotic bacteria widely used in food industry. There are significant differences in adsorption and tolerance of cadmium in different Lactobacillus plantarum strains. Two strains of Lactobacillus plantarum(strongly adsorbed strain CCFM8610 and weakly adsorbed strain CCFM595) with significant differences in cadmium adsorption capacity were screened. The differentially expressed proteins between the two strains were studied by comparative proteomic analysis based on isobaric tags for relative and absolute quantification(iTRAQ). The results showed that totally 1 690 proteins were identified in the two strains, 109 abundant differentially expressed proteins and89 proteins were exclusively detected in CCFM8610. Through functional annotation, metabolic pathway reconstruction and interaction network analysis of these differentially expressed proteins, the possible mechanisms of cadmium adsorption and tolerance of Lactobacillus plantarum CCFM8610 are as follows: it has a unique energy-saving metabolic pattern, which is characterized by the lower overall level of TCA cycle pathway proteins; expresses more proteins involved in cell wall and extracellular polymer synthesis, thus enhancing the isolation and adsorption of cadmium ions; it has stronger metal efflux transporting capacity, such as cadmium efflux pump such as cadmium also has higher abundance in CCFM8610;CCFM8610 shows higher expression of proteins involved in biosynthesis of hydrophobic amino acids, thus improving surface hydrophobicity of the strain and meanwhile upregulating certain amino acids to bind Cd to tackle Cd toxicity.
引文
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[8]MOSA K A,SAADOUN I,KUMAR K,et al.Potential biotechnological strategies for the cleanup of heavy metals and metalloids[J].Frontiers in Plant Science,2016,7:303.
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[11]RAMADOSS J,MAGNESS R R.Alcohol-induced alterations in maternal uterine endothelial proteome:A quantitative iTRAQ mass spectrometric approach[J].Reproductive Toxicology,2012,34(4):538-544.
[12]TRIBOULET S,AUDE-GARCIA C,CARRI魬REM,et al.Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses[J].Molecular&Cellular Proteomics,2013,12(11):3108-3122.
[13]MA J,SHENG H,LI X,et al.iTRAQ-based proteomic analysis reveals the mechanisms of siliconmediated cadmium tolerance in rice(Oryza sativa)cells[J].Plant Physiology and Biochemistry,2016,104:71-80.
[14]HALTTUNEN T,COLLADO M C,EL-NEZAMI H,et al.Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution[J].Letters in Applied Microbiology,2008,46(2):160-165.
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[16]QIAO J,WANG J,CHEN L,et al.Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp.PCC 6803[J].Journal of Proteome Research,2012,11(11):5286-5300.
[17]BAI J,YANG X,DU R,et al.Biosorption mechanisms involved in immobilization of soil Pb by Bacillus subtilis DBM in a multi-metal-contaminated soil[J].Journal of Environmental Sciences,2014,26(10):2056-2064.
[18]ZHAI Q,TIAN F,WANG G,et al.The cadmium binding characteristics of a lactic acid bacterium in aqueous solutions and its application for removal of cadmium from fruit and vegetable juices[J].Rsc Advances,2016,6(8):5990-5998.
[19]BEVERIDGE T J,MURRAY R G.Sites of metal deposition in the cell wall of Bacillus subtilis[J]Journal of Bacteriology,1980,141(2):876-87.
[20]LAMBERT P A,HANCOCK I C,BADDILEY JInfluence of alanyl ester residues on the binding of magnesium ions to teichoic acids[J].Biochemical Journal,1975,151(3):671-676.
[21]PAL A,PAUL A.Microbial extracellular polymeric substances:Central elements in heavy metal bioremediation[J].Indian Journal of Microbiology,2008,48(1):49.
[22]NIES D H.Efflux-mediated heavy metal resistance in prokaryotes[J].FEMS Microbiology Reviews,200327(2/3):313-339.
[23]SOLOVIEVA I,ENTIAN K D.Investigation of the yvgW Bacillus subtilis chromosomal gene involved in Cd2+ion resistance[J].FEMS Microbiology Letters,2002,208(1):105-109.
[24]SHARMA R,RENSING C,ROSEN B P,et al The ATP hydrolytic activity of purified ZntA,a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli[J].Journal of Biological Chemistry,2000,275(6):3873-3878.
[25]SNAVELY M D,FLORER J B,MILLER C G,et al.Magnesium transport in Salmonella typhimurium:28Mg2+transport by the CorA,MgtA,and MgtBsystems[J].Journal of Bacteriology,1989,171(9):4761-4766.
[26]SERRANO L M,MOLENAAR D,WELS M,et al Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1[J].Microbial Cell Factories,2007,6(1):29.
[27]IZAWA S,MAEDA K,MIKI T,et al.Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae[J].Biochemical Journal,1998,330(2):811-817.
[28]TANG W,XING Z,LI C,et al.Molecular mechanisms and in vitro antioxidant effects of Lactobacillus plantarum MA2[J].Food Chemistry,2017,221:1642-1649.
[29]DI CAGNO R,DE ANGELIS M,LIMITONE A,et al.Response of Lactobacillus helveticus PR4 to heat stress during propagation in cheese whey with a gradient of decreasing temperatures[J].Appl Environ Microbiol,2006,72(7):4503-4514.
[30]WU H,SOLER-GARCIA A,JERSE A E.Astrain-specific catalase mutation and mutation of the metal-binding transporter gene mntC attenuate Neisseria gonorrhoeae in vivo but not by increasing susceptibility to oxidative killing by phagocytes[J].Infection and Immunity,2009,77(3):1091-1102.
[31]TREMAROLI V,WORKENTINE M L.,WELJIE AM,et al.Metabolomic investigation of the bacterial response to a metal challenge[J].Appl Environ Microbiol,2009,75(3):719-728.
[32]PIETERSE B.Transcriptome analysis of the lactic acid and NaCl-stress response of Lactobacillus plantarum[J].Wur Wageningen Ur,2006,626:1-6.
[33]CRONAN J J,LAPORTE D.Tricarboxylic acid cycle and glyoxylate bypass[J].EcoSal Plus,2005,1(2):79-99
[34]MUJAHID M,PRASUNA M L,SASIKALA C,et al.Integrated metabolomic and proteomic analysis reveals systemic responses of Rubrivivax benzoatilyticus JA2 to aniline stress[J].Journal of Proteome Research,2014,14(2):711-727.
[35]MYKYTCZUK N C S,TREVORS J T,LEDUC LG,et al.Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[J].Progress in Biophysics and Molecular Biology,2007,95(1/2/3):60-82.
[36]MOORE C M,GABALLA A,HUI M,et al.Genetic and physiological responses of Bacillus subtilis to metal ion stress[J].Molecular Microbiology,2010,57(1):27-40.
[37]SHARMA S S,DIETZ K J.The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress[J].Journal of Experimental Botany,2006,57(4):711-726.
[38]SMITH D S,BELL R A,KRAMER J R.Metal speciation in natural waters with emphasis on reduced sulfur groups as strong metal binding sites[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2002,133(1/2):65-74.
[39]KAST P,GRISOSTOMI C,CHEN I A,et al.Astrategically positioned cation is crucial for efficient catalysis by chorismate mutase[J].Journal of Biological Chemistry,2000,275(47):36832-36838.
[40]WEGRZYN G,WEGRZYN A.Stress responses and replication of plasmids in bacterial cells[J].Microbial Cell Factories,2002,1(1):2.
[2]LIU X,TIAN G,JIAN D G,et al.Cadmium(Cd)distribution and contamination in Chinese paddy soils on national scale[J].Environmental Science&Pollution Research International,2016,23(18):1-12.
[3]FRIBERG L,NORDBERG G F,VOUK V BHandbook on the toxicology of metals[M].Amsterdam:Elsevier/North-Holland Biomedical Press,1979:124-128.
[4]RUSHHOFT C C.The possibilities of disposal of radioactive wastes by biological treatment methods[J].Sewage Works Journal,1949,21(5):877-883.
[5]TABAK H H,LENS P,VAN HULLEBUSCH E D,et al.Developments in bioremediation of soils and sediments polluted with metals and radionuclides-1.Microbial processes and mechanisms affecting bioremediation of metal contamination and influencing metal toxicity and transport[J].Reviews in Environmental Science and Bio/Technology,2005,4(3):115-156.
[6]HALTTUNEN T,SALMINEN S,TAHVONEN RRapid removal of lead and cadmium from water by specific lactic acid bacteria[J].International Journal of Food Microbiology,2007,114(1):30-35.
[7]ZHAI Q,WANG G,ZHAO J,et al.Protective Effects of Lactobacillus plantarum CCFM8610 against acute cadmium toxicity in mice[J].Applied and Environmental Microbiology,2013,79(5):1508-1515.
[8]MOSA K A,SAADOUN I,KUMAR K,et al.Potential biotechnological strategies for the cleanup of heavy metals and metalloids[J].Frontiers in Plant Science,2016,7:303.
[9]VEGLIO F,BEOLCHINI F.Removal of metals by biosorption:A review[J].Hydrometallurgy,1997,44(3):301-316.
[10]ZHAI Q,TIAN F,WANG G,et al.The cadmium binding characteristics of a lactic acid bacterium in aqueous solutions and its application for removal of cadmium from fruit and vegetable juices[J].Rsc Advances,2016,6(8):5990-5998.
[11]RAMADOSS J,MAGNESS R R.Alcohol-induced alterations in maternal uterine endothelial proteome:A quantitative iTRAQ mass spectrometric approach[J].Reproductive Toxicology,2012,34(4):538-544.
[12]TRIBOULET S,AUDE-GARCIA C,CARRI魬REM,et al.Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses[J].Molecular&Cellular Proteomics,2013,12(11):3108-3122.
[13]MA J,SHENG H,LI X,et al.iTRAQ-based proteomic analysis reveals the mechanisms of siliconmediated cadmium tolerance in rice(Oryza sativa)cells[J].Plant Physiology and Biochemistry,2016,104:71-80.
[14]HALTTUNEN T,COLLADO M C,EL-NEZAMI H,et al.Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution[J].Letters in Applied Microbiology,2008,46(2):160-165.
[15]LEE J Y,PAJARILLO E A,KIM M J,et al.Proteomic and transcriptional analysis of Lactobacillus johnsonii PF01 during bile salt exposure by iTRAQshotgun proteomics and quantitative RT-PCR[J].Journal of Proteome Research,2012,12(1):432-443.
[16]QIAO J,WANG J,CHEN L,et al.Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp.PCC 6803[J].Journal of Proteome Research,2012,11(11):5286-5300.
[17]BAI J,YANG X,DU R,et al.Biosorption mechanisms involved in immobilization of soil Pb by Bacillus subtilis DBM in a multi-metal-contaminated soil[J].Journal of Environmental Sciences,2014,26(10):2056-2064.
[18]ZHAI Q,TIAN F,WANG G,et al.The cadmium binding characteristics of a lactic acid bacterium in aqueous solutions and its application for removal of cadmium from fruit and vegetable juices[J].Rsc Advances,2016,6(8):5990-5998.
[19]BEVERIDGE T J,MURRAY R G.Sites of metal deposition in the cell wall of Bacillus subtilis[J]Journal of Bacteriology,1980,141(2):876-87.
[20]LAMBERT P A,HANCOCK I C,BADDILEY JInfluence of alanyl ester residues on the binding of magnesium ions to teichoic acids[J].Biochemical Journal,1975,151(3):671-676.
[21]PAL A,PAUL A.Microbial extracellular polymeric substances:Central elements in heavy metal bioremediation[J].Indian Journal of Microbiology,2008,48(1):49.
[22]NIES D H.Efflux-mediated heavy metal resistance in prokaryotes[J].FEMS Microbiology Reviews,200327(2/3):313-339.
[23]SOLOVIEVA I,ENTIAN K D.Investigation of the yvgW Bacillus subtilis chromosomal gene involved in Cd2+ion resistance[J].FEMS Microbiology Letters,2002,208(1):105-109.
[24]SHARMA R,RENSING C,ROSEN B P,et al The ATP hydrolytic activity of purified ZntA,a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli[J].Journal of Biological Chemistry,2000,275(6):3873-3878.
[25]SNAVELY M D,FLORER J B,MILLER C G,et al.Magnesium transport in Salmonella typhimurium:28Mg2+transport by the CorA,MgtA,and MgtBsystems[J].Journal of Bacteriology,1989,171(9):4761-4766.
[26]SERRANO L M,MOLENAAR D,WELS M,et al Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1[J].Microbial Cell Factories,2007,6(1):29.
[27]IZAWA S,MAEDA K,MIKI T,et al.Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae[J].Biochemical Journal,1998,330(2):811-817.
[28]TANG W,XING Z,LI C,et al.Molecular mechanisms and in vitro antioxidant effects of Lactobacillus plantarum MA2[J].Food Chemistry,2017,221:1642-1649.
[29]DI CAGNO R,DE ANGELIS M,LIMITONE A,et al.Response of Lactobacillus helveticus PR4 to heat stress during propagation in cheese whey with a gradient of decreasing temperatures[J].Appl Environ Microbiol,2006,72(7):4503-4514.
[30]WU H,SOLER-GARCIA A,JERSE A E.Astrain-specific catalase mutation and mutation of the metal-binding transporter gene mntC attenuate Neisseria gonorrhoeae in vivo but not by increasing susceptibility to oxidative killing by phagocytes[J].Infection and Immunity,2009,77(3):1091-1102.
[31]TREMAROLI V,WORKENTINE M L.,WELJIE AM,et al.Metabolomic investigation of the bacterial response to a metal challenge[J].Appl Environ Microbiol,2009,75(3):719-728.
[32]PIETERSE B.Transcriptome analysis of the lactic acid and NaCl-stress response of Lactobacillus plantarum[J].Wur Wageningen Ur,2006,626:1-6.
[33]CRONAN J J,LAPORTE D.Tricarboxylic acid cycle and glyoxylate bypass[J].EcoSal Plus,2005,1(2):79-99
[34]MUJAHID M,PRASUNA M L,SASIKALA C,et al.Integrated metabolomic and proteomic analysis reveals systemic responses of Rubrivivax benzoatilyticus JA2 to aniline stress[J].Journal of Proteome Research,2014,14(2):711-727.
[35]MYKYTCZUK N C S,TREVORS J T,LEDUC LG,et al.Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[J].Progress in Biophysics and Molecular Biology,2007,95(1/2/3):60-82.
[36]MOORE C M,GABALLA A,HUI M,et al.Genetic and physiological responses of Bacillus subtilis to metal ion stress[J].Molecular Microbiology,2010,57(1):27-40.
[37]SHARMA S S,DIETZ K J.The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress[J].Journal of Experimental Botany,2006,57(4):711-726.
[38]SMITH D S,BELL R A,KRAMER J R.Metal speciation in natural waters with emphasis on reduced sulfur groups as strong metal binding sites[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2002,133(1/2):65-74.
[39]KAST P,GRISOSTOMI C,CHEN I A,et al.Astrategically positioned cation is crucial for efficient catalysis by chorismate mutase[J].Journal of Biological Chemistry,2000,275(47):36832-36838.
[40]WEGRZYN G,WEGRZYN A.Stress responses and replication of plasmids in bacterial cells[J].Microbial Cell Factories,2002,1(1):2.