摘要
金黄色葡萄球菌(Saphylococcus aureus)是人类和某些动物的病原菌之一,其致病力强弱主要取决于产生的毒素和侵袭性酶的能力,其中金黄色葡萄球菌耐热性肠毒素,是引起人类食物中毒和葡萄球菌胃肠炎的主要原因,因此,这方面的研究已成为科学家们普遍关注的重要课题。本文以主要产B型肠毒素的金黄色葡萄球菌S6菌株为研究对象,采用X—射线衍射、扫描电镜、原子力显微镜、分子振动光谱(红外、激光拉曼)、圆二色谱、荧光光谱等现代分析技术表征金黄色葡萄球菌B型肠毒素(staphylococcal enterotoxin B,简称SEB)的聚集态结构、三维立体形貌、分子链构象及蛋白质二级结构组成。同时着重研究了肠毒素在稀溶液状态和外界环境改变时的构象特征及可能的去折叠过程,尝试用蛋白质构象理论揭示肠毒素耐热的根本原因,寻找其耐热的可能优势构象,建立研究蛋白质构象与生物学活性相关性的新方法。此外,还从酸奶发酵菌株和金黄色葡萄球菌的混合培养特性出发,研究了乳酸菌及代谢产物对金黄色葡萄球菌的抑制作用及其在抑制过程中热效应的微量变化,从热化学的角度探讨其作用机制。主要研究结果如下:
1.金黄色葡萄球菌B型肠毒素的分离纯化
采用玻璃纸覆盖琼脂平板法培养金黄色葡萄球菌,首先利用羧甲基纤维素阳离子CM-32柱层析,对B型肠毒素进行初步分离富集,然后依据筛分原理按分子量大小经Sephadex G-75凝胶进一步纯化,得到电泳纯B型肠毒素,得率为26.8%,紫外吸收光谱测得其特征吸收峰为276.6nm,SDS-PAGE凝胶电泳分析其相对分子质量为2.8×10~4,与文献报道值一致。
2.金黄色葡萄球菌B型肠毒素的结构表征
利用X-射线衍射、扫描电镜、原子力显微镜、傅里叶变换红外光谱、激光拉曼光谱等现代分析手段对金黄色葡萄球菌B型肠毒素的聚集态结构、分子链形态,二级结构等进行了表征。结果表明,SEB为一种单纯蛋白,其聚集态包含结晶区和无定形区,结晶度为37.8%,其分子聚集在一起,形态主要为片状和平板状,具有一定的规整性。原子力显微镜首次观察到SEB在稀溶液状态下的单分子链长度约为1500nm,分子链宽度约为20～40nm,分子链高0.6nm。SEB的傅里叶变换红外光谱和激光拉曼光谱分析表明,SEB的二级结构单元主要为β—折叠和α—螺旋,无规卷曲含量较少,侧链C—C—S—S—C—C构型为反式—扭曲—反式。
3.金黄色葡萄球菌B型肠毒素的稀溶液构象及去折叠作用
研究大分子的溶液构象和蛋白质的去折叠作用是近来揭示蛋白质生物活性及其与高级结构相关性的重要切入点。金黄色葡萄球菌B型肠毒素水溶液(浓度为
Staphylococcus aureus is one of the major human and animal pathogens that produce a wide range of toxins and a group of enzymes contributing to their ability to cause diseases. Staphylococcal enterotoxins (SEs), a family of ten major serological types of heat stable enterotoxins, are a key cause of staphylococcal food poisoning and gastroenteritis in human. Staphylococcal food poisoning is the second most common food poisoning events caused by bacterial pathogens. In this paper, Staphylococcus aureus S6 producing staphylococcal enterotoxin B was studied. On the basis of purified SEB, aggregation structure, the molecule chain morphology and secondary structural parameters of SEB were investigated with the advanced techniques, such as X-ray diffraction, scan electron microscopy, atomic force microscopy, fourier transform infrared spectrum, laser raman spectrum, circular dichroism spectrometer and fluorescence spectrum. The characteristic conformation and possible unfolding process of SEB in dilute solution and different conditions were emphatically studied. Furthermore, the inhibition of Staphylococcus aureus by lactic acid bacteria and their metabolizing substance was studied. The inhibiting mechanism of nisin on Staphylococcus aureus was also studied from the point of microcalorimetry. The main results from this study are as follows:1. The isolation and purification of SEBStaphylococcal enterotoxin B was obtained in the relative yield of 26.8% by the method of cellophane covered agar plate and electrophoretic homogeneity from the culture of Staphylococcus aureus. The major purification procedure involved ion-exchange chromatography with CM-cellulose and gel filtration with Sephadex G-200. The molecular weight of purified SEB was determined as 2.8 × 10~4 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the characteristic absorbance peak on UV spectrum was 276.6nm, which is consistent with that in the reported literature.2. Structure characterization of SEBThe aggregation structure, the molecule chain morphology and secondary structural parameters of SEB were studied by modern analytic techniques, such as X-ray diffraction,
scan electron microscopy, atomic force microscopy > fourier transform infrared spectrum, and laser raman spectrum.The results showed that there was the formation of crystallizing field in SEB with crystallinity of 37.8%. The SEB molecule was conglomerated with the shape of sheet and plate. The molecular chain conformation of SEB was observed directly by AFM, and its length, width and height of molecular chain was 1500nm, 20~40nm and 0.6nm respectively. The secondary structural unit of SEB was mainly composed of P -sheets and a -helixs with few random coils. The side chain conformation of C—C—S — S —C—C was trans-twist-trans.3. The conformation and unfolding of SEB in the dilute solutionThe secondary structure of SEB derived from the far-UV circular dichroic spectra were composed of 22.6% a -helixs,40.4% P -sheets,19.2% turns and 17.7% random coils in water solution at the concentration of 0.02mol/L (25 °C). Within the range of 50°C and 121 "C, the content of a -helixs gradually descended from 22.6% at 25 °C to 9.6% at 95 °C with the increase of temperature, and a -helixs conformation of SEB completely disappeared at 121 °C. In opposition to the change of a -helixs content in SEB, the content of 3-sheets remained comparatively stable, with the exception at 75 °C the content of P -sheets was 8.5%. The content of P -sheets was up to 39.9% at 121 "C (more than 30.2% at other temperatures). The results showed that the change of temperature almost could not induce unfolding of SEB, and P -sheet was the major unit to maintain the structural stability and microbio-activity of SEB. The Laser Raman Spectrum of SEB obtained by heat treatment at 121 °C indicated that the characteristic vibration of a -helixs and S-S linkage was disappeared, but still remained vibrating band of P -sheets at 1672cm"1. These results proved that SEB was a heat stable proteins, and provided theoretical foundation for this property. There have been no reports on similar results previously.The effect of SDS, urea and nisin on the conformation of SEB was also studied by circular dichroism spectrometer and fluorescence spectrum. Though the content of a -helixs in the secondary structure of SEB decreased, the content of P -sheets and random coils increased, the major conformation was still P -sheets after the treatment of SDS and nisin. This means that SDS and nisin did not make SEB deactivated. On the other hand, the content of a -helixs in (he secondary structure of SEB remarkably increased, while P -sheets and random coils disappeared, and turned to P -turn after the treatment of urea. This means that urea did unfolding action on SEB, and confirms the reported prediction that urea treatment was not suitable for the detection of heat-denatured enterotoxins in the
thermally processed foods.It was indicated in fluorescence spectrum experiments that the tryptophane residue in SEB turned to hydrophobic environment and fluorescence quantum yield and relative intensity decreased with the increase of urea concentrations (0~6mol/L). On the other hand, the tryptophane residue in SEB turned to hydrophilic environment and the relative intensity of SEB increased with the concentration increase of SDS and nisin. In addition, 30% ethanol and 30% glycerol made the intrinsic fluorescence intensity of SEB an increase of 29.9% and 461.0% respectively, but the maximum emission wavelength remained unchanged, which showed the same effect as SDS did. SEB showed faintly effect on the fluorescence spectra of nisin with little increasing of relative intensity, and the maximum emission wavelength of nisin remained unchanged.4. Preparation of the antibody against staphylococcal entrotoxin B andestablishment of indirect competition ELISAfor SEB detectionThe immune protocol of little dose and long period was adopted to immune Balb/c mouse with injecting SEB by multi dots and multi times. When the mouse serum titer was appropriate, the Balb/c mouse was injected with a non-antibody-secreting myeloma cell P2/0 to induce ascites formation, which was then extracted for the preparation of antibody. It had a potential application when the titer of antibody measured by indirect ELIS A achieved l:105.An indirect competition ELISA for detecting SEB was established by homemade SEB and anti-SEB antibody in this study. The detection limit of indirect ELISA assay established in this study was lng/ml with a detection concentration range of 1—10Jng/mL and correlation coefficient r=0.9951.The recovery of artificially contaminated sample was 94.5%.5.The inhibitory effects of lactic acid bacteria and their metabolicsubstances on Staphylococcus aureusThe growth of staphylococcus aureus was inhibited by a low pH> the occurring of anti-microbial substances and action of nutritional competition among microorganisms when Staphylococcus aureaus, Lactobacillus bulgaricus and Streptococcus thermophilus were grown by a commixture culture. Lactic acid, acetic acid and H2O2 showed inhibitory effect on the growth of staphylococcus aureus with positive correlation to their concentrations. The inhibition of lactic acid and acetic acid on Staphylococcus aureus was stronger than that of inorganic acid (hydrochloric acid), and they both showed obvious
inhibitions on the growth of Staphylococcus aureus at pH 3.5. The inhibition of nisin on the growth of Staphylococcus aureus was correlative with its concentration, and the correlation could be expressed as y=0.8025x+5.12, R2=0.9939.6. Effect of nisin on the growth of Staphylococcus aureus by amicrocalorimetric methodA novel microcalorimetric technique was applied to evaluate the biological effect of nisin on the growth of Staphylococcus aureus. The physiological, biochemical and metabolic characteristics of Staphylococcus aureus changed in the presence of nisin. The results showed that the growth rate constant of Staphylococcus aureus decreased and the generation time prolonged with the increasing of nisin concentration. The relationship between the growth rate constant (k) and the concentration of nisin (c) is k=0.0377-3.988xl0"4c, with a correlation coefficient of -0.9971. Half inhibitory concentration of nisin on the growth of Staphylococcus aureus was 48.05IU/mL, the critical inhibitory concentration was 94.53IU/mL. The growth rate constant, the generation time, the half inhibitory concentration and the critical inhibitory concentration obtained by microcalorimetric method which could be used as a quantitative index for evaluating the inhibitory effect of nisin on microorganisms, which could be also applied for evaluating the inhibitory effect of other bacteriocins. There have been no reports on similar results previously.7. Aspects on the ecology of Staphylcococcus aureus in yoghurtThe initial inoculating amount of Staphylococcus aureus in raw milk was investigated on its existent ability in yoghurt. No Staphylcococcus aureus and its products thermonuclease and staphylococcal enterotoxin B were detected in yoghurt during the fermentation and storage when its initial inoculation in raw milk were 1.8 X 10 cfu/mL and 1.8 X102 cfu/mL. When the initial inoculation in raw milk were 8.5 X103, 2.7 X104, 2.7 X105 and 1.8 X 106cfu/mL, the amount of Staphylcococcus aureus increased gradually during the process of fermentation, while it decreased during the stage of storage, and at the same time the thermonuclease was positive and the content of SEB were 0> 6.5, 38.5 an 77.9ng/mL respectively. This means that the growth of Staphylcococcus aureus and the production of enterotoxins were not effectively inhibited by the low pH and metabolic substances when the raw milk was contaminated with large amount of the bacteria. There was a hazard to cause staphylococcal food poisoning if these foods were consumed.
引文
1.中华人民共和国中家标准:GB4789.10-94食品微生物学检验.金黄色葡萄球菌检验
2.王丽哲,晋怀峰,宫树权.金黄色葡萄球菌污染水产食品的研究.食品科学,2001,22(5):78-80
3.李献,马家斌.金黄色葡萄球菌在蘑菇罐头中生长繁殖与产毒规律的研究.四川食品工业科技,1994,13(2):24-26
4.孟昭赫.食品卫生检验方法注解微生物学部分.北京:人民卫生出版社,1990,102
6.胡东良,林雁春,刘佩红.乳及乳制品中金黄色葡萄球菌的分布及其理化特性研究.食品科学,1996a,17(1):69-73
7.胡东良,刘清河,刘佩红.肉品中金黄色葡萄球菌的检测方法及其污染状况的研究.肉品卫生,1996b,(6):3-7
8.胡东良,品川邦汛,刘佩红.动物性食品中金黄色葡萄球菌产肠毒素的研究.中国食品卫生杂志,1996c,(3):9-11
9.袁育康.医学免疫学与病原生物学.北京医科大学出版社.2000.
10.苏世彦.食品微生物检验手册.北京:中国轻工业出版社,1998:88
11.舒泉,陈建华,秦生巨,封幼玲,徐凌云,陶炳根.金黄色葡萄球菌质粒分型的研究.中华微生物学和免疫学杂志,1992,12(3):148-153
12.候安新,刘义,黄伟国,薛智,屈松生.钇离子及其阳离子卟啉配合物对金黄色葡萄球菌的相互作用.化学学报,2003,61(9):1382-1387
13.雷祚荣,Reiser R F, Bergdoll M S.层析等电聚焦法提纯葡萄球菌肠毒素D.生物化学杂志,1990,6(2):117-122
14. A C Papageorgiou, K R Acharya, R. Shapiro, E F Passalacqua, R D Brehm,H S Tranter.Crystal structure of the superantigen enterotoxin C_2 from Staphylococcus aureus reveals a zinc-binding site. Structure, 1995,3:769-779
15. Alber G, Hammer D,Fleischer B.Relationship between enterotoxic and T lymphocyte stimulating activity of staphylococcal enterotoxin B. J Immunol, 1990, 144:4501-4506
16. Anastassios C P, HOward S T, Acharya K R. Crystal structure of microbial superantigen staphylococcal enterotoxin B at 1.5 (?) resolution: implications for superantigen recognition by MHC class Ⅱ molecules and T-cell receptors. J Mol Biol,1998,277(1):61-79
17. Arbuthnott J P. Staphylococcal toxins in human disease.J Appl Bacteriol Supplement,1990,101S-107S.
18. Aveaa R M., Bergdoll M. S.Purification and some physiochemical properties of enterotoxin C.Biochemistry,1967,6:1474-1480
19. Bayles K W,Iandolo J J. Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin DJ Bacteriol, 1989,171:4799-4806
20. Bergdoll M S.Analytical methods for staphylococcus aureus.Int J Food Microbiol,1990,10:91-100
21. Bergdoll M S. Enterotoxins.In Easmon C S F, Adlam C, Staphylococci and staphylococcal infections. Academic Press, Ltd., London, United Kingdom. 1983,559-598
22. Bergdoll M.Staphylococcal intoxications.In H Riemann,F L Bryan,Food-borne infections and intoxications.New York:Academic Press.1979,443-494
23. Bergdoll M S, Robbins R N, Weiss K, Borja C R, Huang I Y, Chu F S. The staphylococcal enterotoxins: similarities. Contrib Microbiol Immunol,1973,1:390-396
24. Betley M J, Mekalanos J J.Nucleotide sequence of the type A staphylococcal enterotoxin gene.J. Bacteriol, 1988, 170:34-41
25. Bohach G A, Dinges M M, Mitchell D T, Ohlendorf D H, Schlievert P M. Exotoxins, In Crossley K B, Archer G L.The staphylococci in human disease. Churchill Livingstone, Inc., New York, N.Y. 1997,83-111
26. Buelow R,Ohehir R E,Scherifels R.Localization of the immunologic activity in the superantigen staphylococcal enterotoxin B using truncated recombinant fusion proteins J Immunol,1993,148:1-5
27. Casman E P,M S Bergdoll,J Robinson.Designation of Staphylococcal enterotoxins. J Bacteriol, 1963,85:715-716
28. Christopher L J , Saleem A K.Nucleotide sequence of the enterotoxin B gene from Staphylcoccus aureus. J.Bacteriol,1986,166(1):29-33
29. Couch J L,Soltis M T, Betley M J.Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.J Bacteriol, 1988,170:2954-2960
30. Crawley G J,Black J N,Gray I.Clinical chemistry of staphylococcal enterotoxin poisoning in monkeys.Appl Microbiol,1966,14(3):445-450
31. Czop J K, Bergdoll M S.Staphylococcal enterotoxin synthesis during the exponential, transitional, and stationary growth phases.Infect Immun, 1974, 9:229-235
32. Dinges M M,0rwin P M,Schlievert P M.Exotoxins of staphylococcus aureus.Clinical Microbiol Reviews,2000,13:16-34
33. Ende I A, Terplan G, Kickhofen B, Hammer D K. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1 Appl Environ Microbiol. 1983,46(6):1323-1330
34. Esald S.Evaluation of enzyme-linked immunosorben assay(ELISA) for detection of staphylococcal enterotoxin in foods. Int J Food Microbiol,l988,6:141-153
35. Evenson M 1,Hinds M W,Bernstein R S.Bergdoll M S.Estimation of human dose of staphylococcal enterotoxin A from a large outbreak of staphylococcal food poisoning involving chocolate milk.Int J Food Microbiol,1988,7-311-316
36. Fey H, Pfister H, Muller C. Simple production of staphylococcal enterotoxins with chromatofocusing and isoelectrofocusing in flat bed gels.Zentralbl Veterinarmed 5,1984 ,31(7):508-17
37. Freaet J I E,McCoy F, Strong M. Purification of type B staphylococcal enterotoxin J Bacteriol ,1963,86:1308-1313
38. Haines W C, Harmon L G.Effect of selected lactic acid bacteria on growth of Staphylococcus aureus and production of enterotoxin.Appl Microbiol, 1973, 25(3):436-441
39. Harris T O,Grossman D,Kappler J W,Marrack P,Rich R R,Betley M J.Lack of complete correlation between emetic and T-cell stimulatory activities of staphylococcal enterotoxins.Infect Immun,1993,61:3175-3183
40. Hovde C J,Hackett S P,Bohach G A. Nucleotide sequence of the staphylococcal enterotoxin C_3 gene:sequence comparison of all three type C staphylococcal enterotoxins.Mol Gen Genet,1990,220:329-333
41. Huang I Y,Bergdoll M S.The primary structure of staphylococcal enterotoxin B. III.The cyanogens bromide peptides of reduced and aminoethylated enterotoxin B and the complete amino acid sequence J Biol Chem, 1970,245:3518-3525
42. Irwin M J,Hudson K R,Fraser J D.Enterotoxin residues determining T-cell receptor V β binding specificity Nature ,1992,359:841-845
43. Johnson H W,Russell J K,Pontzer C H.Super-antigens in human diseas.Sci Amer,1992,266(4):92-101
44. Jones LW.Effect of butterfat on inhibition of Staphylococcus aureus by nisin.Can J Microbiol ,1974,20(9):1257-1260
45. Lee R Y,Silverman G J,Munsey D T.Growth and entertoxin A production by staphylococcus aureus in precooked bacon in the intermediate moisture range J Food Sci ,1981,46:1687-1692
46. Lei Z, Reiser R F, Bergdoll M S.Chromatofocusing in the purification of staphylococcal enterotoxin D J Clin Microbiol,1988,26(6):1236-1237
47. Margaret I, Dohnalek M I, Marth E H. Growth and production of enterotoxin A by Staphylococcus aureus in cream J Dairy Sci,1989 ,72(9):2266-2275
48. Meyrand A, Boutrand L S, Ray G S.Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.J Appl Microbiol,1998,85(3):537-544
49. Middlebrook J L, Spero L, Argos P.The secondary structure of staphylococcal enterotoxins A, B and C. Biochim Biophys Acta, 1980,621(2):233-240
50. Morse S A,Mah R A,Dobrogosz W J.Regulation of staphylococcal enterotoxin B.J Bacteriol, 1969,98:4-9
51. M Sundstrom, L Abrahmsen, P Antonsson, K Mehindate, W Mourad,M Dohlsten.The crystal structure of staphylococcal enterotoxin type D reveals Zn~2+-mediated homodimerization. EMBO J,1996,15:6832-6840
52. Munoz P A, Warren J R, Noelken M E.Beta structure of aqueous staphylococcal enterotoxin B by spectropolarimetry and sequence-based conformational predictions. Biochemistry, 1976,15(21):4666-4671
53. Naomi B, Avraham R. Staphylococcal enterotoxins. Int J Food Microbiol, 2000, 61(1): 1-10
54. Notermans S,Heuvelman C J.Combined effect of water activity.pH and suboptimal temperature on growth and enterotoxin production of staphylococcu aureus. J Food Sci,1983,48:1832-1835
55. Nunez M, Bautista L, Medina M, Gaya P. Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese J Appl Bacteriol,l988,65(1):29-34
56. Orwin P M,Leung D Y,Donahue H L,Novick R P.Schlievert P M.Biochemical and biological properties of staphylococcal enterotoxin K. Infect Immunol, 2001, 69: 360-366
57. Otero A, Garcia M C, Garcia M L. Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2,during the manufacture and storage of Burgos cheese J Appl Bacteriol,1988,64(2): 117-122
58. Park C E,Akhtar M,Rayman M K.Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit(TECRA) for detection of staphylococcal enterotoxins in foods.Appl Environ Microbiol,1992,58:2509-2512
59. Pazakova J,Turek P,Laciakova A.The survival of Staphylococcus aureus during the fermentation and storage of yoghurt. J Appl Bacteriol, 1997,82:659-662
60. Peter F.Appendix 1.Rapid methods for detecting food borne pathogens.Bacteriological Analytical Manual Online(FDA-CFSAN),2001,l
61. Schad E M,Zaitseva I,Zaitsev V N.Crystal structure of the superantigen staphylococcal enterotoxin type A.EMBOJ ,l995,14:3292-3296
62. Singh B R Evenson M L, Bergdoll M S. Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy.Biochemistry, 1988,27(24):8735-8741
63. Singh B R, Betley M J. Comparative structural analysis of staphylococcal enterotoxins A and E.J Biol Chem, 1989,264(8):4404-4411
64. Smith J L,Buchanan R I,Palumbo S A.Effect of food environment on staphylococcal enterotoxin synthesis:a reviewJ Food Prot, 1983,46:545-555
65. Stewart C M,Cole M B,Legan J D.Modeling the growth boundary of staphylococcus aureus for risk assessment purposes. J Food Prot,2001,64(l):51-57
66. Su Y C, Wong A C L.Identification and purification of a new staphylococcal enterotoxin, H Appl Environ Microbiol ,1995,61(4):1438-1443
67. Su Y C, Wong A C L.Current perspectives on detection of staphylococcal enterotoxins. J Food Protect, 1997,60: 195-202
68. Swaminathan S ,Furey W ,Pletcher J.Crystal structure of staphylococcal enterotoxin B, a superantigen Nature (London),1992,359:801-806
69. Tibana A K,Rayman M A.Szabo R.Thermal stability of staphylococcal enterotoxins A,B and C in a buffered system J .Food Protect,1987,50:239-242
70. Troller J A,Stinson J V.Influence of water activity on the production of extracellular enzymes by staphylococcus aureus .Appl Environ Microbiol,1978,35(3):521-526
71. Wieneke A A.Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int J Food Microbiol,1991,14:305-312