Mycolactone: A polyketide toxin from mycobacterium ulcerans required for virulence

Science

Volumn 283, Issue 5403, 1999, Pages 854-857

  George, Kathleen M ^a   Chatterjee, Delphi ^b   Gunawardana, Geewananda ^c   Welty, Diane ^a   Hayman, John ^d   Lee, Richard ^e   Small, P L C ^a  

^a NATIONAL INSTITUTES OF HEALTH   (United States)

^b Department of Environmental and Radiological Health Sciences   (United States)

^c ABBOTT LABORATORIES   (United States)

^d BOX HILL HOSPITAL   (Australia)

^e NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL TOXIN;

ANIMAL EXPERIMENT; ANIMAL MODEL; ARTICLE; BACTERIAL VIRULENCE; CELL CULTURE; CELL CYCLE; CLINICAL FEATURE; CYTOTOXICITY; HUMAN TISSUE; MITOSIS INHIBITION; NONHUMAN; PATHOGENESIS; PRIORITY JOURNAL; SKIN DISEASE;

ANIMALS; BACTERIAL TOXINS; CELL CYCLE; CHROMATOGRAPHY, HIGH PRESSURE LIQUID; CHROMATOGRAPHY, THIN LAYER; FEMALE; GUINEA PIGS; L CELLS (CELL LINE); MASS SPECTROMETRY; MICE; MYCOBACTERIUM INFECTIONS, ATYPICAL; MYCOBACTERIUM ULCERANS; NECROSIS; SKIN; SKIN DISEASES, BACTERIAL; VIRULENCE;

ANIMALIA; BACTERIA (MICROORGANISMS); CAVIA; MURINAE; MYCOBACTERIUM ULCERANS;

EID: 0033524996     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5403.854     Document Type: Article

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9. For toxin production, M. ulcerans 1615 (Trudeau Collection Strain, Lake Saranac, NY) were grown in Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol and 10% oleic acid, albumin, dextrose, and catalase enrichment (Difco) and incubated at 32°C. We harvested cells at late exponential growth phase by passage through a 0.22-μm filter. Bacteria were scraped off the filter, weighed, and extracted by stirring with chloroform and methanol (2:1) for 4 hours. We removed the bacterial debris by centrifugation, and added 0.2 volume of water to the supernatant. The organic phase was removed and dried in a rotoevaporator, and the remaining lipids were resuspended in ice-cold acetone to precipitate phospholipids. The ASL fraction was run on silica TLC to separate individual lipid components by using chloroform, methanol, and water (90:10:1) as a solvent system. Lipid bands were scraped off, and lipids were eluted in the organic solvent in which they were run. These lipids were dried down, weighed, and resuspended in acetone at 4°C. Under these conditions, mycolactone did not lose biological activity for 6 weeks.
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25. We thank L. Barker, B. Caughey, T. Schwan, and L. Katz for critical reading of this report. We express particular appreciation to D. Brooks for assistance with flow cytometry, C. Favara for her skillful preparation of sections for histopathology, and P. Brennan for his insight and support. Animal experiments were conducted under guidelines of the Animal Care and Use Committee at Rocky Mountain Laboratories.