分枝杆菌霉菌酸的提取和纯化

Christian M. Dupont; Laurent Kremer

doi:10.21769/BioProtoc.1265

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Peer-reviewed

Extraction and Purification of Mycobacterial Mycolic Acids

分枝杆菌霉菌酸的提取和纯化

CD Christian M. Dupont

LK Laurent Kremer email

发布: 2014年10月20日第4卷第20期 DOI: 10.21769/BioProtoc.1265 浏览次数: 13875

评审: Fanglian HeElizabeth Libby

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

May 2014

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Abstract

Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a critical role in the architecture and impermeability of the cell envelope, hence the natural resistance of mycobacteria to most antibiotic treatments. They are also key determinants of virulence in pathogenic species, including Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis. In addition, they are known as the primary target of several first-line and second-line antitubercular drugs. Thus, the unique enzymes involved in the mycolic acid biosynthetic pathway represent an attractive reservoir of targets for future chemotherapy whose developments are particularly warranted in the context of multi-drug-resistant and extensively-drug-resistant strains of M. tuberculosis. Herein, we describe a protocol to extract the mycolic acids from mycobacteria. Purification of the various subspecies may be particularly useful for subsequent structural studies involving mass spectrometry or NMR. The qualitative and quantitative biochemical characterization of the mycolic acid pattern by thin layer chromatography can be used to address how drugs alter mycolic acid biosynthesis (Alahari et al., 2007, Hartkoorn et al., 2012), to study the phenotypes of genetically modified mutants affected in this metabolic pathway (Bhatt et al., 2007) or to unravel new mycolic acid regulatory mechanisms (Vilcheze et al., 2014). The same protocol can be applied to all mycobacteria, including environmental and pathogenic species.

Keywords: Mycobacterium (结核分枝杆菌)

Mycolic acid (分枝菌酸)

Cell wall (细胞壁)

Thin layer chromatography (薄层色谱法)

Lipid (脂质)

Materials and Reagents

Mycobacterial cultures including Mycobacterium tuberculosis, BCG, M. smegmatis and M. abscessus but can be extended to any other species.
Tyloxapol (C₁₇H₃₀O₄) (Sigma-Aldrich, catalog number: T8761 )
Glycerol (C₃H₈O₃, 99.5%) (Euromedex, catalog number: 50405 )
Potassium diphosphate (K₂HPO₄) (Merck KGaA, catalog number: 1.05104 )
L-asparagine monohydrate (C₄H₁₀N₂O₄, ≥99%) (Sigma-Aldrich, catalog number: A8381 )
Citric acid (C₆H₈O₇, 99%) (Sigma-Aldrich, catalog number: C0759 )
Ferric ammonium citrate (C₇H₁₃FeNO₇) (Sigma-Aldrich, catalog number: F5879 )
Zinc sulfate heptahydrate (ZnSO₄^.7H₂O, ≥99%) (Sigma-Aldrich, catalog number: Z4750 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
Tetrabutyl ammonium hydroxyde (TBAH) (C₁₆H₃₇NO, 40% solution) (Sigma-Aldrich, catalog number: 178780 )
Dichloromethane (CH₂Cl₂, ≥99.8%) (Carlo Erba, catalog number: 412622000 )
Iodomethane (CH₃I, 99%) (Sigma-Aldrich, catalog number: I850-7 )
Diethylether (CH₃CH₂OCH₂CH₃, 99.5%) (Carlo Erba, catalog number: 447521 )
Hexane [CH₃(CH₂)₄CH₃, pure isomeric mixture] (Carlo Erba, catalog number: 339852 )
Ethyl acetate (CH₃COOC₂H₅, 99.9%) (Carlo Erba, catalog number: 448251 )
Molybdophosphoric acid hydrate (H₅Mo₁₂O₄₁P) (Sigma-Aldrich, catalog number P7390 )
Rhodamine 6G (C₂₈H₃₁ClN₂O₃, Dye content ~95 %) (Sigma-Aldrich, catalog number R4127 )
Middlebrook 7H9 broth (Difco, catalog number: 271310 )
Sauton’s media (see Recipes)

Equipment

Aluminium Silica gel TLC plates 60F₂₅₄ (Merck KGaA, catalog number: 1.05554.0001 )
Plastic Silica gel TLC plates 60F₂₅₄ (Merck KGaA, catalog number: 1.05735.0001 )
Round-bottom borosilicate glass tubes (Corning, catalog number: 99449-16 ) with phenolic screw caps (Corning, catalog number: 9998-15 )
10 µl capillary tubes Ringcaps (Dutscher Scientific, catalog number: 0 90512 )
Spectrophotometer
75 cm² tissue culture flasks (Sarstedt, catalog number: 83.1813 )
37 °C incubator or 30 °C incubator for growing mycobacteria such as M. marinum
Notes: Atypical mycobacteria and BCG should be handled according to institutional standards of practice for biosafety whereas M. tuberculosis cultures should be handled in biosafety level 3 facility.
50 ml conical Corning CentriStart tubes (Corning, catalog number: 430828 )
15 ml conical Corning CentriStart tubes (Corning, catalog number: 430790 )
Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Prolabo, model: HR340 with the 15 ml and 50 ml adaptors)
pH meter
Ultrasonic waterbath (Bandelin Sonorex, model: RK102H )
Stuart Rotator SB3
Stuart block heater (Stuart, model: SBH200D )
TLC developing tank
Zymark TurboVap evaporator (Biotage, catalog number: C133718 )
Handheld UV lamp VL-6-LC 365 nm/254 nm
1800W Heat gun (Power Performance, model: PHG1800T2 )
Reagent Sprayer, all glass, with 100 ml Erlenmeyer flask (CAMAG, catalog number: 022.6100 )
TLC Spray Cabinet (CAMAG, catalog number: 0 22.623 0)

Procedure

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中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Dupont, C. M. and Kremer, L. (2014). Extraction and Purification of Mycobacterial Mycolic Acids . Bio-protocol 4(20): e1265. DOI: 10.21769/BioProtoc.1265.
Alahari, A., Trivelli, X., Guérardel, Y., Dover, L. G., Besra, G. S., Sacchettini, J. C., Reynolds, R. C., Coxon, G. D. and Kremer, L. (2007). Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria. PLoS One 2(12): e1343.