JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Traduction automatique
Please note that all translations are automatically generated. Click here for the English version.
Biochimie

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Published: May 15, 2019
doi:
10.3791/59538
, ,
1Department of Microbiology and Molecular Genetics,Michigan State University, 2Department of Physiology,Michigan State University

Summary

该方法为研究将来自复杂宿主来源的外源脂肪酸结合到细菌膜中提供了一个框架,特别是金黄色葡萄球菌。为此,介绍了从鸡蛋蛋黄中浓缩脂蛋白颗粒的方案,以及利用质谱法对细菌磷脂进行随后脂肪酸分析的介绍。

Abstract

金黄色葡萄球菌和其他革兰氏阳性病原体将环境中的脂肪酸结合到膜磷脂中。在感染期间,大多数外源脂肪酸存在于宿主脂蛋白颗粒中。宿主脂肪酸的储存库以及细菌从脂蛋白颗粒中提取脂肪酸的机制仍存在不确定性。在这项工作中,我们描述了从鸡蛋蛋黄中浓缩低密度脂蛋白(LDL)颗粒的协议,并确定LD是否作为S.aureus的脂肪酸储存库。该方法利用无偏脂质学分析和鸡低密度脂蛋白,是探索低密度脂蛋白与细菌相互作用的有效经济模型。采用高分辨率/精确质谱法和串联质谱法对低密度脂蛋白中外源脂肪酸的S.氨基酸整合进行分析,从而对细菌的脂肪酸成分进行表征膜和无偏的鉴定在接触低密度脂蛋白时在细菌膜脂质中产生的脂肪酸的新组合。这些先进的质谱技术通过揭示并入磷脂中的特定外源脂肪酸,为脂肪酸的加入提供了无与伦比的视角。此处概述的方法适用于其他细菌病原体和复杂脂肪酸的替代来源的研究。

Introduction

耐甲氧西林(MRSA)是卫生保健相关感染的主要原因,相关的抗生素耐药性是一个相当大的临床挑战1,2,3。因此,制定新的治疗策略是重中之重。革兰氏阳性病原体的一个有前途的治疗策略是抑制脂肪酸合成,这是膜磷脂生产的要求,在S.aureus中,包括磷脂甘油(PG)、裂解-PG和心肌利平4。在细菌中,脂肪酸的产生通过脂肪酸合成II途径(FASII)5发生,这与真核对应物有很大不同,使得FASII成为抗生素开发5,6个有吸引力的靶点。.FASII抑制剂主要针对FabI,这是脂肪酸碳链伸长化所需的一种酶。FabI抑制剂三氯生被广泛用于消费品和医疗用品8,9。几家制药公司正在开发额外的FabI抑制剂,用于治疗10、11、12、13、14 ,15,16,17,18,19,20,21,22,23 ,24,25,26.然而,许多革兰氏阳性病原体,包括S.aureus,能够清除外源脂肪酸的磷脂合成,绕过FASII抑制27,28,29。因此,FASII抑制剂的临床潜力是辩论的,因为我们对宿主脂肪酸的来源和病原体从宿主27、28中提取脂肪酸的机制的认识存在相当大的差距。为了弥补这些差距,我们开发了一种无偏脂质学分析方法,以监测脂蛋白颗粒中外源脂肪酸与S.aureus膜磷脂的合并情况。

在败血症期间,宿主脂蛋白颗粒是血管内宿主衍生脂肪酸的潜在来源,因为大多数宿主脂肪酸都与颗粒30相关。脂蛋白由亲水外壳组成,由磷脂和蛋白质组成,这些蛋白质包裹着甘油三酯和胆固醇酯的疏水性核心31。四大类脂蛋白 – 奇洛美亚、极低密度脂蛋白、高密度脂蛋白和低密度脂蛋白 (LDL) – 由宿主生产,并充当脂质运输工具,提供脂肪酸和胆固醇。通过血管的宿主细胞。低密度脂蛋白富含酯化脂肪酸,包括甘油三酯和胆固醇酯31。我们之前已经证明,高度纯化的人类低密度脂蛋白是PG合成的外源脂肪酸的可行来源,从而为FASII抑制剂旁路32提供了机制。纯化人体低密度脂蛋白在技术上可能具有挑战性且耗时,而纯化人类低密度脂蛋白的商业来源在日常使用或进行大规模细菌筛选时成本高昂。为了解决这些限制,我们修改了从鸡蛋蛋黄中浓缩低密度脂蛋白的程序,鸡蛋蛋黄是脂蛋白颗粒33的丰富来源。我们已经成功地使用非目标,高分辨率/准确的质谱和串联质谱监测将人类LDL衍生脂肪酸结合到S.aureus32膜中。与先前报告的方法不同,这种方法可以量化三种主要葡萄球菌磷脂类型的单个脂肪酸异构体。油酸(18:1)是存在于所有宿主脂蛋白颗粒中的不饱和脂肪酸,很容易并入S.aureus磷脂29,30,32。金黄色葡萄球菌不能合成油酸29;因此,磷脂结合的油酸的数量建立在葡萄球菌膜29中存在宿主脂蛋白衍生脂肪酸。这些磷脂物种可以通过这里描述的最先进的质谱法来识别,在脂肪酸来源存在的情况下,为培养的S.aureus的膜组成提供了前所未有的分辨率。感染期间遭遇。

Protocol

注:以下从鸡蛋蛋黄中浓缩低密度脂蛋白颗粒的协议来自穆萨等人2002年33。 1. 制备鸡蛋黄以浓缩低密度脂蛋白颗粒 用70%乙醇溶液清洗壳,使两个大鸡蛋消毒,并晾干。 使用 70% 乙醇溶液对鸡蛋分离器进行消毒,并允许空气干燥。将鸡蛋分离器连接到中型烧杯的唇上。 将每个鸡蛋分别裂入鸡蛋分离器中,让白蛋白流入烧杯。完整的蛋黄将由分?…

Representative Results

图1说明了从鸡蛋蛋黄中浓缩低密度脂蛋白的协议。这个过程首先用盐水稀释整个蛋黄,并将蛋黄固体从含有LDL的可溶性或血浆部分中分离出,称为颗粒(图1)33。血浆馏分的LDL含量通过+30-40 kDaβ-活蛋白的沉淀进一步丰富(图2)33。蛋白质带在140、80、65、60和15 kDa的存在与低密?…

Discussion

Aureus将外源脂肪酸加入其膜磷脂27,32,43。使用外源性脂肪酸的磷脂合成绕过了FASII抑制,但也改变了膜27、32、44的生物物理特性。虽然将外源性脂肪酸纳入革兰氏阳性病原体磷脂是有据可查的,但在宿主脂肪酸储存库的身份和三种主要葡萄球菌磷脂类型?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢哈默实验室的成员对手稿的批判性评价和对这项工作的支持。科罗拉多大学医学院的亚历克斯·霍斯威尔博士亲切地提供了AH1263。密歇根州立大学的克里斯·沃特斯博士实验室提供了试剂。这项工作得到了美国心脏协会16SDG30170026的资助和密歇根州立大学提供的启动基金的支持。

Materials

Ammonium sulfate Fisher BP212R-1 ≥99.5% pure
Cell culture incubator Thermo MaxQ 6000
Centrafuge Thermo 75-217-420 Sorvall Legen XTR, rotor F14-6×250 LE
Costar assay plate Corning 3788 96 well
Filter paper Schleicher & Schuell 597
Large chicken egg N/A N/A Common store bought egg
Microplate spectrophotometer BioTek Epoch 2
NaCl Sigma S9625
S. aureus strain AH1263 N/A N/A Provided by Alex Horswill of the University of Colorado
Dialysis tubing Pierce 68700 7,000 MWCO
Tryptone Becton, Dickison and Company 211705
0.5 mm zirconium oxide beads Next Advance ZROB05
Bullet Blender Next Advance BBX24B
Methanol (LC-MS grade) Fisher A4561
Chloroform (reagent grade) Fisher MCX10559
Isopropanol (LC-MS grade) Fisher A4611
Dimyristoyl phosphatidylcholine Avanti Polar Lipids 850345C-25mg
Ammonium bicarbonate Sigma 9830 ≥99.5% pure
Ammonium formate Sigma 70221-25G-F
Xcalibur software Thermo Scientific OPTON-30801
LTQ-Orbitrap Velos mass spectrometer Thermo Scientific high resolution/accurate mass MS
Agilent 1260 capillary HPLC Agilent
SpeedVac Vacuum Concentrators Thermo Scientific
DOWNLOAD MATERIALS LIST

References

  1. Noskin, G. A., et al. National trends in Staphylococcus aureus infection rates: impact on economic burden and mortality over a 6-year period (1998-2003). Clinical Infectious Diseases. 45 (9), 1132-1140 (2007).
  2. Noskin, G. A., et al. The burden of Staphylococcus aureus infections on hospitals in the United States: an analysis of the 2000 and 2001 Nationwide Inpatient Sample Database. Archives of Internal Medicine. 165 (15), 1756-1761 (2005).
  3. Laible, B. R. Antimicrobial resistance: CDC releases report prioritizing current threats. South Dakota medicine. 67 (1), 30-31 (2014).
  4. Zhang, Y. M., Rock, C. O. Membrane lipid homeostasis in bacteria. Nature Reviews Microbiology. 6 (3), 222-233 (2008).
  5. Zhang, Y. M., White, S. W., Rock, C. O. Inhibiting bacterial fatty acid synthesis. Journal of Biological Chemistry. 281 (26), 17541-17544 (2006).
  6. Sohlenkamp, C., Geiger, O. Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiology Reviews. 40 (1), 133-159 (2016).
  7. Schiebel, J., et al. Staphylococcus aureus FabI: inhibition, substrate recognition, and potential implications for in vivo essentiality. Structure. 20 (5), 802-813 (2012).
  8. Heath, R. J., Li, J., Roland, G. E., Rock, C. O. Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. Journal of Biological Chemistry. 275 (7), 4654-4659 (2000).
  9. Heath, R. J., Yu, Y. T., Shapiro, M. A., Olson, E., Rock, C. O. Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. Journal of Biological Chemistry. 273 (46), 30316-30320 (1998).
  10. Park, H. S., et al. Antistaphylococcal activities of CG400549, a new bacterial enoyl-acyl carrier protein reductase (FabI) inhibitor. Journal of Antimicrobial Chemotherapy. 60 (3), 568-574 (2007).
  11. Schiebel, J., et al. Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor. Journal of Biological Chemistry. 289 (23), 15987-16005 (2014).
  12. Yum, J. H., et al. In vitro activities of CG400549, a novel FabI inhibitor, against recently isolated clinical staphylococcal strains in Korea. Antimicrobial Agents and Chemotherapy. 51 (7), 2591-2593 (2007).
  13. Kaplan, N., et al. Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective antistaphylococcal FabI inhibitor. Antimicrobial Agents and Chemotherapy. 56 (11), 5865-5874 (2012).
  14. Karlowsky, J. A., Kaplan, N., Hafkin, B., Hoban, D. J., Zhanel, G. G. AFN-1252, a FabI inhibitor, demonstrates a Staphylococcus-specific spectrum of activity. Antimicrobial Agents and Chemotherapy. 53 (8), 3544-3548 (2009).
  15. Ross, J. E., Flamm, R. K., Jones, R. N. Initial broth microdilution quality control guidelines for Debio 1452, a FabI inhibitor antimicrobial agent. Antimicrobial Agents and Chemotherapy. 59 (11), 7151-7152 (2015).
  16. Hunt, T., Kaplan, N., Hafkin, B. Safety, tolerability and pharmacokinetics of multiple oral doses of AFN-1252 administered as immediate release (IR) tablets in healthy subjects. Journal of Chemotherapy. 28 (3), 164-171 (2016).
  17. Hafkin, B., Kaplan, N., Hunt, T. L. Safety, tolerability and pharmacokinetics of AFN-1252 administered as immediate release tablets in healthy subjects. Future Microbiology. 10 (11), 1805-1813 (2015).
  18. Flamm, R. K., Rhomberg, P. R., Kaplan, N., Jones, R. N., Farrell, D. J. Activity of Debio1452, a FabI inhibitor with potent activity against Staphylococcus aureus and coagulase-negative Staphylococcus spp., including multidrug-resistant strains. Antimicrobial Agents and Chemotherapy. 59 (5), 2583-2587 (2015).
  19. Yao, J., Maxwell, J. B., Rock, C. O. Resistance to AFN-1252 arises from missense mutations in Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI). Journal of Biological Chemistry. 288 (51), 36261-36271 (2013).
  20. Tsuji, B. T., Harigaya, Y., Lesse, A. J., Forrest, A., Ngo, D. Activity of AFN-1252, a novel FabI inhibitor, against Staphylococcus aureus in an in vitro pharmacodynamic model simulating human pharmacokinetics. Journal of Chemotherapy. 25 (1), 32-35 (2013).
  21. Parsons, J. B., et al. Perturbation of Staphylococcus aureus gene expression by the enoyl-acyl carrier protein reductase inhibitor AFN-1252. Antimicrobial Agents and Chemotherapy. 57 (5), 2182-2190 (2013).
  22. Kaplan, N., et al. In vitro activity (MICs and rate of kill) of AFN-1252, a novel FabI inhibitor, in the presence of serum and in combination with other antibiotics. Journal of Chemotherapy. 25 (1), 18-25 (2013).
  23. Kaplan, N., Garner, C., Hafkin, B. AFN-1252 in vitro absorption studies and pharmacokinetics following microdosing in healthy subjects. European Journal of Pharmaceutical Sciences. 50 (3-4), 440-446 (2013).
  24. Banevicius, M. A., Kaplan, N., Hafkin, B., Nicolau, D. P. Pharmacokinetics, pharmacodynamics and efficacy of novel FabI inhibitor AFN-1252 against MSSA and MRSA in the murine thigh infection model. Journal of Chemotherapy. 25 (1), 26-31 (2013).
  25. Karlowsky, J. A., et al. In vitro activity of API-1252, a novel FabI inhibitor, against clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy. 51 (4), 1580-1581 (2007).
  26. Yao, J., et al. A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome. Antimicrobial Agents and Chemotherapy. 60 (7), 4264-4273 (2016).
  27. Brinster, S., et al. Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens. Nature. 458 (7234), 83-86 (2009).
  28. Balemans, W., et al. Essentiality of FASII pathway for Staphylococcus aureus. Nature. 463 (7279), E3-E4 (2010).
  29. Parsons, J. B., Frank, M. W., Subramanian, C., Saenkham, P., Rock, C. O. Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors. Proceedings of the National Academy of Sciences of the United States of America. 108 (37), 15378-15383 (2011).
  30. Abdelmagid, S. A., et al. Comprehensive profiling of plasma fatty acid concentrations in young healthy Canadian adults. PLoS One. 10 (2), e0116195 (2015).
  31. Feingold, K. R., Grunfeld, C. Introduction to Lipids and Lipoproteins. Endotext. , (2000).
  32. Delekta, P. C., Shook, J. C., Lydic, T. A., Mulks, M. H., Hammer, N. D. Staphylococcus aureus utilizes host-derived lipoprotein particles as sources of exogenous fatty acids. Journal of Bacteriology. 200 (11), (2018).
  33. Moussa, M., Marinet, V., Trimeche, A., Tainturier, D., Anton, M. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. Theriogenology. 57 (6), 1695-1706 (2002).
  34. Breil, C., Abert Vian, M., Zemb, T., Kunz, W., Chemat, F. Bligh and Dyer and Folch Methods for Solid-Liquid-Liquid Extraction of Lipids from Microorganisms. Comprehension of Solvatation Mechanisms and towards Substitution with Alternative Solvents. International journal of molecular sciences. 18 (4), (2017).
  35. Lydic, T. A., Busik, J. V., Reid, G. E. A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids. Journal of Lipid Research. 55 (8), 1797-1809 (2014).
  36. Bowden, J. A., Bangma, J. T., Kucklick, J. R. Development of an automated multi-injection shotgun lipidomics approach using a triple quadrupole mass spectrometer. Lipids. 49 (6), 609-619 (2014).
  37. Haimi, P., Uphoff, A., Hermansson, M., Somerharju, P. Software tools for analysis of mass spectrometric lipidome data. Analytical Chemistry. 78 (24), 8324-8331 (2006).
  38. Hewelt-Belka, W., et al. Comprehensive methodology for Staphylococcus aureus lipidomics by liquid chromatography and quadrupole time-of-flight mass spectrometry. Journal of Chromatography A. 1362, 62-74 (2014).
  39. Jolivet, P., Boulard, C., Beaumal, V., Chardot, T., Anton, M. Protein components of low-density lipoproteins purified from hen egg yolk. Journal of Agricultural and Food Chemistry. 54 (12), 4424-4429 (2006).
  40. Bylesjö, M., et al. OPLS discriminant analysis: combining the strengths of PLS-DA and SIMCA classification. Journal of Chemometrics. 20 (8-10), 341-351 (2006).
  41. Noble, R. C., Cocchi, M. Lipid metabolism and the neonatal chicken. Progress in Lipid Research. 29 (2), 107-140 (1990).
  42. Cherian, G., Holsonbake, T. B., Goeger, M. P. Fatty acid composition and egg components of specialty eggs. Poultry Science. 81 (1), 30-33 (2002).
  43. Parsons, J. B., Frank, M. W., Rosch, J. W., Rock, C. O. Staphylococcus aureus Fatty Acid Auxotrophs Do Not Proliferate in Mice. Antimicrobial Agents and Chemotherapy. 57 (11), 5729-5732 (2013).
  44. Sen, S., et al. Growth-Environment Dependent Modulation of Staphylococcus aureus Branched-Chain to Straight-Chain Fatty Acid Ratio and Incorporation of Unsaturated Fatty Acids. PLoS One. 11 (10), e0165300 (2016).
  45. Wang, M., Huang, Y., Han, X. Accurate mass searching of individual lipid species candidates from high-resolution mass spectra for shotgun lipidomics. Rapid Communications in Mass Spectrometry. 28 (20), 2201-2210 (2014).
  46. Peti, A. P. F., Locachevic, G. A., Prado, M. K. B., de Moraes, L. A. B., Faccioli, L. H. High-resolution multiple reaction monitoring method for quantification of steroidal hormones in plasma. Journal of Mass Spectrometry. 53 (5), 423-431 (2018).
  47. Neves, M. M., Heneine, L. G. D., Henry, M. Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia. 66 (3), 769-777 (2014).
  48. Liu, P. V., Hsieh, H. C. Inhibition of Protease Production of Various Bacteria by Ammonium Salts – Its Effect on Toxin Production and Virulence. Journal of Bacteriology. 99 (2), 406 (1969).
  49. Suller, M. T., Russell, A. D. Triclosan and antibiotic resistance in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy. 46 (1), 11-18 (2000).
  50. Yao, C. H., Liu, G. Y., Yang, K., Gross, R. W., Patti, G. J. Inaccurate quantitation of palmitate in metabolomics and isotope tracer studies due to plastics. Metabolomics. 12, (2016).

Tags

Keywords: Lipoprotein ParticlesChicken Egg YolkBacterial PathogenFatty Acid IncorporationMembrane PhospholipidsLipidomic AnalysisStaphylococcus AureusAmmonium SulfateDialysis
check_url/fr/59538?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
Delekta, P. C., Lydic, T. A., Hammer, N. D. Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids. J. Vis. Exp. (147), e59538, doi:10.3791/59538 (2019).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image