摘要
研究表明,多种芽胞杆菌都可以产生环状脂肽类物质来抑制植物病原真菌的生长。本研究从水稻根部分离出一株有较广抑菌谱、能产生脂肽的解淀粉芽胞杆菌WH1,并通过两步萃取、凝胶过滤层析等方法从WH1发酵液中纯化到了其产生的脂肽。经薄层层析、红外图谱、电喷雾质谱等分析后发现其是一种surfactin类的脂肽,将其命名为WH1fungin。本研究还对WH1fungin的抑菌机制进行了研究。
在抑菌试验中发现用100μg/ml的WH1fungin处理真菌能使真菌坏死,同时也检测到了核浓缩、DNA断裂、活性氧(ROS)积累、磷酯酰丝氨酸(PS)外翻、caspase活性上升等凋亡特征。
研究表明ROS在真菌凋亡中起到非常重要的作用,实验中用脯氨酸、抗坏血酸或还原型谷胱甘肽等活性氧清除剂处理后检测到活性氧积累减少,但磷脂酰丝氨酸外翻并不呈相应的减少,表明WH1fungin诱导的细胞凋亡不完全依赖于活性氧积累。
实验中发现经WH1fungin处理后真菌的葡聚糖合酶活性有明显下降。而葡聚糖合酶活性的降低会导致胼胝质合成量的减少,使新形成细胞没有足够的胼胝质来合成细胞壁,而细胞壁不完整的细胞更加容易发生细胞凋亡。
实验中还利用亲和层析分离到了与WH1fungin相互作用蛋白,经肽指纹图谱分析其为线粒体F-ATPase,在后续的检测中也验证了经WH1fungin处理后的真菌细胞的ATPase活性明显下降。在用FITC标记的WH1fungin处理细胞后能检测到其结合到线粒体膜上,验证了WH1fungin能作用于线粒体。寡霉素是线粒体FoF1-ATPase的抑制剂,WH1fungin处理的真菌细胞在加入寡霉素后凋亡现象更为显著,进一步验证WH1fungin能通过作用于线粒体ATPase来发挥促真菌凋亡的作用。
综上所述,WH1产生的脂肽在低浓度时可通过诱导凋亡的方式表现出抗真菌活性,这种凋亡可能是通过抑制葡聚糖合酶活性及作用于线粒体ATPase完成的。在自然生态中芽胞杆菌产生的脂肽并不足以导致细胞膜的穿孔,但却可诱导凋亡的发生,由此推断,WH1fungin诱导的细胞凋亡可能在解淀粉芽胞杆菌对真菌的拮抗作用中起重要的作用。
Some species of Bacillus could produce lipopeptides for controlling plant pathogenic fungi. In this study, we isolated a strain of B. amyloliquefaciens, called as WH1, from the rice root. WH1 could produce lipopeptide, WHlfungin, to inhibit several plant pathogenic fungi. WH1fungin could be isolated from the culture of WH1 by extraction and gel filtration, and characterized as an anti-fungal lipopeptide (surfactin) by thin layer chromatography, infrared spectrum, Tricine-SDS-PAGE and mass spectrum. We also studied on the anti-fungal mechanism of it.
After treated by WH1fungin, it was found that WH1fungin could directly kill some fungal cells by eliciting pores on membrane, while WHlfungin also could induce apoptosis. Classic apoptotic markers such as nucleus condensation, ROS accumulation, PS externalization, DNA strand breaks and high caspase-like activity were detectable.
Because ROS is a key factor for fungal apoptosis, further investigation was done for elucidating the role of ROS in apoptotic fungal cells. When fungal cells were treated by WHlfungin, antioxidants such as ascorbic acid, proline and reduced glutathione (GSH) were used for cleaning ROS respectively. It was found that ascorbic acid had no effect on the ROS, while proline and GSH could significantly inhibit ROS accumulation in WHlfungin-treated fungal cells. Unfortunately, clean of ROS by proline and GSH couldn't inhibit the relevant PS externalization in fungal cells.
It was reported that several cyclic lipopeptides could inhibit the activity of fungal glucane synthase, resulting in a synthesis decrease of callose. New generated fungal cells without enough callose for forming cell wall were easily found in apoptosis. In this study we found the activity of fungal glucane synthase was obviously decreased, and correspondent apoptotic markers could be detected in WHlfungin-treated fungal cells.
In order to detect the targeted proteins of WH1fungin, we used WH1fungin conjugated Sepharose 4B to isolate proteins in fungal cells. A purified protein was collected after isolation by this affinity chromatography, and was identified as a mitochondrial F-ATPase by peptide mass fingerprinting. After treated by FITC labeled WH1fungin, it was showed that WH1fungin could bind to the mitochondrial memebrane of fungal cells. After treated by WH1fungin, ATPase activity was significantly lower than control groups. It's known that oligomycin is an inhibitor of mitochondrial FoF1-ATPase of fungal cells. In this study more ROS and PS externalization was observed in fungal cells when oligomycin was used for treating fungal cells together with WHlfungin. All of these data verified that WH1fungin could interact with ATPase on the mitochondrial membrane
As a conclusion, low level of WH1fungin produced by WH1 could suppress the fungal glucane synthase, inhibit the mitochondrial ATPase, and then induce apoptosis in fungal cells. Normally Bacillus couldn't produce enough lipopeptide to elicit pores on fungal membrane in the natural environment, so we deduced that induction of apoptosis might be the common anti-fungal mechanism of Bacillus in the natural habitat.
引文
1.陈华,王丽,袁成凌等.高效液相色谱—电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物.色谱,2008,26(3):343—347
2.陈亮,浦冠勤.化学防治与生物防治在害虫综合防治中的作用.中国蚕业,2008,29(4):84—86,90
3.陈中义,张杰,黄大防.植物病害生防芽胞杆菌抗菌机制与遗传改良研究.植物病理学报,2003,33(2):97—103
4.崔艳红,黄现青.生物表面活性剂—表面活性素.生物技术,2006,16(5):91—94
5.管珺,胡永红,杨文革等.蜡样芽胞杆菌防治植物病虫害的研究进展.现代农药,2007,6(4):7—10
6.侯红漫,靳艳,金美芳等.环脂肽类生物表面活性剂结构、功能及生物合成.微生物学通报,2006,33(5):122—128
7.姜倩,林琳,汪天虹.研究细胞凋亡的新模式生物—酵母.生物化学与生物物理进展,2008,35(4):361—367
8.李超,伏圣博,刘华玲等.细胞凋亡研究进展.世界科技研究与发展,2007,29(3):45—53
9.刘伯宁,石磊,蒋沁.环脂肽类抗生素研究进展.中国抗生素杂志,2007,32(9):520—524
10.吕莹,张德禄,张宏等.细胞凋亡检测方法研究进展.西北师范大学学报(自然科学版),2007,43(2):82—87
11.丘振宇,王亚琴,许喜林.细胞凋亡的研究.现代食品科技,2007,23 (2):101—104
12.林琳,姜倩,汪天虹.丝状真菌细胞凋亡研究方法.菌物学报,2008,27(3):474—481
13.任争光,张志勇,魏艳敏.芽胞杆菌防治园艺植物病害的研究进展.中国生物防治,2006,22:194—198
14.孙雪文,周金燕,邓洪渊等.有效分离1 kDa分子量环脂肽的电泳方法.生物 技术,2005,15(6):47—49
15.唐娟,张毅,李雷雷.地衣芽胞杆菌应用研究进展.湖北农业科学,2008,47(3):351—354
16.杨福廷.脂肽类生物表面活性剂研究进展.精细化工,2006,23(2):121—125
17.于颖,徐桂花.羊心脏中细胞色素C的提取研究.肉类研究,2009,4:31—34
18.张彦杰,罗俊彩,武燕萍等.生防枯草芽胞杆菌研究进展.生命科学仪器,2009,7:19—23
19.周建华,丛国正,高闪电等.细胞凋亡的研究进展.生物技术通讯,2008,19(2):274—276
20. Arguelles-Arias A, Ongena M, Halimi B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact,2009,8:63
21. Bemheimer A W, Avigad L S. Nature and properties of a cytolytic agent produced by Bacillus subtilis. J Gen Microbiol,1970,61(3):361-369
22. Binder U, Oberparleiter C, Meyer V et al. The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signaling of Aspergillus nidulans. Mol Microbiol, 2010,75(2):294-307
23. Cai J, Jones D P. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome C loss. J Biol Chem,1998,273(19):11401-11404
24. Cao X H, Wang A H, Wang C L et al.. Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. Chem Biol Interact,2010,183,357-362
25. Chen H M and Yan X J. Antioxidant activities of agaro-oligosaccharides with different degrees of polymerization in cell-based system. Biochim Biophys Acta, 2005,1722(1):103-111
26. Chen S R and Dickman M B. Bcl-2 family members localize to tobacco chloroplasts and inhibit programmed cell death induced by chloroplast-targeted herbicides. J Exp Bot,2004,55(4):2617-2623
27. Chen C and Dickman M B. Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii. Proc Natl Acad Sci U S A,2005,102:3459-3464
28. Cheng J, Park T, Chio L, et al. Induction of Apoptosis by Sphingoid Long-Chain Bases in Aspergillus nidulans. Mol Cell Biol,2003,23:163-177
29. Chevtzoff C, Vallortigara J,Averet N et al. The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth. Biochim Biophys Acta,2005,1706:117-125
30. Cory S and Adams J M. The BCL-2 family:Regulators of the cellular life-or-death switch. Nat Rev Cancer,2002,2(9):647-656
31. Deleu M, Paquot M and Nylander T. Effect of Fengycin, a Lipopeptide Produced by Bacillus subtilis, on Model Biomembranes. Biophys J,2008,94:2667-2679.
32. Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochrome C-dependent caspase activation by eliminating IAP inhibition. Cell,2000,102,33-42
33. Elmore S. Apoptosis:A Review of Programmed Cell Death. Toxicol Pathol,2007, 35:495-516
34. Enari M, Sakahira H, Yokoyama H, et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature,1998,391:43-50
35. Feignier C, Besson F and Michel G. Studies on lipopeptide biosynthesis by Bacillus subtilis:isolation and characterization of iturin-, surfactin+mutants. FEMS Microbiol Lett,1995,127:11-15
36. Garrido C, Galluzzi L, Brunet M, et al. Mechanism of cytochrome C release from mitochondria. Cell Death Differ.2006,13:1423-1433
37. Grangemard I, Wallach J, Maget-Dana R, et al. Lichenysin:A more efficient cation chelator than surfactin. Appl biochem biotech,2001,93(3):199-210
38. Green D R. Apoptotic pathways:Paper wraps stone blunts scissors. Cell.2000,102: 1-4
39. Gourlay C W, Du W and Ayscough K R. Apoptosis in yeast-mechanisms and benefits to a unicellular organism. Mol Microbiol,2006,62(6):1515-1521
40. Gourlay C W and Ayscough K R. Actin-induced hyperactivation of the Ras signaling pathway leads to apoptosis in Saccharomyces cerevisiae. Mol Cell Biol,2006, 26(17):6487-6501
41. Hamann A, Brust D and Osiewacz H D. Apoptosis pathways in fungal growth, development and ageing. Trends Microbiol,2008,6:276-283
42. Ito S, Ihara T, Tamura H, et al. Alpha-tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum. FEBS Lett,2007,581:3217-3222
43. Joza N, Susin S A, Daugas E, et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature.2001,410(6828):549-554
44. Kameda Y, Matsui K, Kato H, et al. Antitumor activity of Bacillus natto.3. Isolation and characterization of a cytolytic substance on Ehrlich ascites carcinoma cells in the culture medium of Bacillus natto KMD 1126. Chem Pharm Bull (Tokyo), 1972,20(7):1551-1557
45. Kim S Y, Kim J Y, Kim S H et al. Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression. FEBS Lett,2007,581,865-871
46. Kracht M, Rokos H, Ozel M, et al. Antiviral and hemolytic activities of surfactin isoforms and their methyl seter derivatives. J Antibiot(Tokyo),1999,52(7):613-619
47. Laun P, Pichova A, Madeo F, et al. Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis. Mol Microbiol,2001,39:1166-1173
48. Leiter E, Szappanos H, Oberparleiter C, et al. Antifungal protein PAF severely affects the integrity of the plasma membrane of Aspergillus nidulans and induces an apoptosis-like phenotype. Antimicrob Agents Chemother,2005,49:2445-2453.
49. Lindum P W, Anthoni U, Christophersen C, et al. N-Acyl-L-homoserine lactone autoinducers control production of an extracellular lipopeptide biosurfactant required for swarming motility of Serratia liquefaciens MG1. J Bacteriol,1998,180(23): 6384-6388
50. Madeo F, Frohlich E and Frohlich K U. A yeast mutant showing diagnostic markers of early and late apoptosis. J Cell Biol,1997,139(3):729-734
51. Madeo F, Frohlich E, Ligr M, et al. Oxygen stress:a regulator of apoptosis in yeast. J Cell Biol,1999,145:757-767
52. Madeo F, Engelhardt S, Herker E, et al. Apoptosis in yeast:a new model system with applications in cell biology and medicine. Curr Genet,2002,41:208-216
53. Maget-Dana R and Ptak M. Interactions of Surfactin with Membrane Models. Biophy J,1995,68:1937-1943.
54. Martinez Del Pozo A, Lacadena V, Mancheno J M, et al. The antifungal protein AFP of Aspergillus giganteus is an oligonucleotide/oligosaccharide binding (OB) fold-containing protein that produces condensation of DNA. J Biol Chem,2002,277: 46179-46183.
55. Meisinger C, Pfanner N and Truscott K N. Isolation of yeast mitochondria. Methods Mol Biol,2006,313:33-39
56. Moffitt M C and Neilan B A. The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides. FEMS Microbiol Lett,2000,191:159-167
57. Narasimhan M L, Damsz B, Coca M A, et al. A plant defense response effector induces microbial apoptosis. Mol Cell,2001,8:921-930.
58. Ongena M and Jacques P. Bacillus lipopeptides:versatile weapons for plant disease biocontrol. Trends Microbiol,2008,16:115-125
59. Pozniakovsky A I, Knorre D A, Markova O V et al. Role of mitochondria in the pheromone-and amiodarone-induced programmed death of yeast.2005, J Cell Biol, 2005,168:257-269
60. Riedl S J and Shi Y G. Molecular mechanisms of caspase regulation during apoptosis. Mol Cell Biol,2004,5:897-907
61. Robson D. Programmed cell death in the Aspergilli and other filamentous fungi. Med Mycol,2006,4:109-114
62. Saelens X, Festjens N, Vande Walle L, et al. Toxic proteins released from mitochondria in cell death. Oncogene,2004,23(16):2861-2874
63. Schagger H. Tricine-SDS-PAGE. Nat Protoc,2006.1:16-23
64. Schriever C A, Fernandez C, Rodvold K A, et al. Daptomycin:A novel cyclic lipopeptide antimicrobial. Am J Health-Syst Pharm,2005,62:1145-1158
65. Shirogane T, Fukada T, Muller J M et al. Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Immunity,1999,11(6): 709-719
66. Sloan M E and Wasserman B P. Susceptibility of UDP-glucose:(1,3)-β-glucan synthase to inactivation by phospholipases and trypsin. Plant Physiol,1989,89: 1341-1344
67. Toure Y, Ongena M, Jacques P, et al. Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. JAppl Microbiol,2004,96:1151-1160
68. Tsukagoshi N, Tamura G and Arima K. A novel protoplast-bursting factor (surfactin) obtained from Bacillus subtilis IAM 1213. I. The effects of surfactin on Bacillus megaterium KM. Biochim Biophys Acta,1970,196(2):204-210
69. Vanittanakom N, Loeffler W, Koch U, et al. Fengycin--a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot (Tokyo),1986, 39(7):888-901
70. Vollenbroich D, Pauli G, Ozel M, et al. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl Environ Microbiol,1997,63(1):44-49
71. Wang C L, Ng T B, Yuan F et al. Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2. Peptides,2007,28,1344-1350
72. Weisburg W G, Barns S M, Pelletier D A et al..16S ribosomal DNA amplification for phylogenetic study. JBacteriol,1991,173(2):697-703
73. Wrasidlo W, Mielgo A, Torres V A et al.. The marine lipopeptide somocystinamide A triggers apoptosis via caspase. Proc Natl Acad Sci,2008,105(7):2313-2318
74. Yamamoto S and Harayama S. PCR amp;ification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol.1995,61(3) 1104-1109
75. Zadrag R, Wojnar L, Bartosz G et al. Does yeast shmooing mean a commitment to apoptosis?. Cell Biol Int,2006,30:205-209