Isolation and Analysis of Mycobacterium tuberculosis Mycolic Acids

Catherine Vilchèze1, William R. Jacobs1

1 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10A.3
DOI:  10.1002/9780471729259.mc10a03s05
Online Posting Date:  June, 2007
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Abstract

Mycolic acids are an important constituent of the mycobacterial cell wall. Changes in the structure or composition of mycolic acids have been associated with modification of cell wall permeability and attenuation of pathogenic Mycobacterium tuberculosis strains. This unit describes the isolation of M. tuberculosis mycolic acids and their analysis by either thin‐layer chromatography or high‐performance liquid chromatography. Both techniques have been extensively used to study (1) mycolic acid biosynthesis, (2) the role of mycolic acids in mycobacterial virulence, (3) the effect of antituberculosis drugs targeting the cell wall, and (4) taxonomic purposes.

Keywords: TLC; HPLC; tuberculosis; extraction; MAMEs

     
 
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Table of Contents

  • Basic Protocol 1: Isolation and Analysis of Mycolic Acids by TLC
  • Alternate Protocol 1: Analysis of Mycolic Acids by Two‐Dimensional Silver TLC
  • Basic Protocol 2: Isolation and Analysis of Mycolic Acids by HPLC
  • Alternate Protocol 2: Analysis of Mycolic Acids by HPLC Using a Chlorinated Solvent Elution System
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Analysis of Mycolic Acids by TLC

  Materials
  • M. tuberculosis culture (unit 10.1)
  • [1‐14C]‐acetic acid, sodium salt in ethanol (1.0 mCi/ml)
  • Vesphene
  • 40% aqueous tetrabutylammonium hydroxide solution
  • Methylene chloride (optima‐grade)
  • Methyl iodide
  • 3 N aqueous hydrochloric acid solution
  • Anhydrous sodium sulfate
  • Nitrogen gas
  • Liquid scintillation counting solution
  • Hexanes (HPLC‐grade)
  • Ethyl acetate (HPLC‐grade)
  • 30‐ml PETG media bottles (Fisher)
  • 37°C incubator with shaker
  • 15‐ml conical tubes
  • Pyrex glass tube (13‐mm o.d. × 100‐mm length) with Teflon‐lined screw caps
  • 50° and 100°C heating block
  • Tube rotator (e.g., Glas Col)
  • Cotton‐plugged Pasteur pipets (optional)
  • 4‐ml vial with screw‐thread cap
  • 5‐µl capillary pipets
  • Silica gel 60 F 254 250‐µm glass plates (5 × 10–cm or 10 × 10–cm)
  • TLC tank with lid
  • Autoradiography cassette
  • X‐ray film

Alternate Protocol 1: Analysis of Mycolic Acids by Two‐Dimensional Silver TLC

  Materials
  • 0.6 M aqueous silver nitrate solution
  • Petroleum ether (b.p. 35° to 60°C, ACS‐certified)
  • Anhydrous diethyl ether (ACS‐certified)
  • 125°C oven
  • Additional reagents and equipment for culturing, labeling, saponifying, methylating, and extracting mycolic acids (see protocol 1)

Basic Protocol 2: Isolation and Analysis of Mycolic Acids by HPLC

  Materials
  • M. tuberculosis (unit 10.3)
  • Vesphene
  • 50% (w/v) aqueous potassium hydroxide solution (9 N)
  • Methanol (HPLC‐grade)
  • 6 N aqueous hydrochloric acid solution
  • Chloroform (HPLC‐grade)
  • Anhydrous sodium sulfate
  • Nitrogen gas
  • p‐bromophenacyl ester derivatization kit (Alltech Associates)
  • Potassium bicarbonate
  • 2‐Propanol (HPLC‐grade)
  • 490‐cm2 sterile roller bottle
  • Pyrex glass tube (16‐mm o.d. × 150‐mm length) with Teflon‐lined screw caps
  • 50°, 85°, and 95°C heating blocks
  • Conical funnel
  • 2‐ml screw‐thread vial with cap
  • Reversed‐phase C 18 column (4.6 × 150–mm, 3‐µm particle size)
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Figures

Videos

Literature Cited

   Butler, W.R. and Guthertz, L.S. 2001. Mycolic acid analysis by high‐performance liquid chromatography for identification of Mycobacterium species. Clin. Microbiol. Rev. 14:704‐726.
   Kremer, L. and Besra, G.S. 2005. A waxy tale, by Mycobacterium tuberculosis. In Tuberculosis and the Tubercle Bacillus (S.T. Cole, K. Davis Eisenach, D.N. McMurray, and W.R.J. Jacobs, eds.) pp. 287‐305. ASM Press, Washington, D.C.
   Minnikin, D.E., Kremer, L., Dover, L.G., and Besra, G.S. 2002. The methyl‐branched fortifications of Mycobacterium tuberculosis. Chem. Biol. 9:545‐553.
   Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall, A. 2001. Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a biosafety level 3 laboratory. J. Clin. Microbiol. 39:769‐771.
   Stodola, F.H., Lesuk, A., and Anderson, R.J. 1938. The chemistry of the lipids of Tubercle bacilli liv. The isolation and properties of mycolic acid. J. Biol. Chem. 126:505‐513.
   Takayama, K., Wang, C., and Besra, G.S. 2005. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18:81‐101.
   Zimhony, O., Cox, J.S., Welch, J.T., Vilcheze, C., and Jacobs, W.R. Jr. 2000. Pyrazinamide inhibits the eukaryotic‐like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Nat. Med. 6:1043‐1047.
Internet Resources
   http://www.cdc.gov/nchstp/tb/Laboratory_Services/maps_tagged.pdf
  Mycolic acid pattern standards for HPLC identification of mycobacteria.
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