Abstract
Toward studying the genetics, biosynthesis, and roles in the pathogenesis of the dominant surface glycopeptidolipid antigens of Mycobacterium avium, rough colony variants of M. avium serovar 2 were picked, cultured in quantity, and their lipid composition examined. Two of the rough (Rg) variants, Rg-3 and Rg-4, were devoid of glycopeptidolipids or any more elemental structures and thus were similar to those described previously. Two others, Rg-0 and Rg-1, each contained two novel lipopeptides, devoid of any of the carbohydrate substituents that confer serotypic activity on the glycopeptidolipids. The application of gas chromatography, fast atom bombardment-mass spectrometry and 1H NMR to lipopeptide I established the structure C32:2-beta-OH-fatty acyl-D-Phe-D-allo-Thr-D-Ala-L-alaninol. Lipopeptide II represented a minor variation of this structure: C32:1-beta-OH-fatty acyl-D-Phe-D-allo-Thr-D-Ala-L-alaninol. These newly discovered lipopeptides are identical to the fatty acyl-tripeptide-amino alcohol "core" component of the glycopeptidolipids of the M. avium complex, and thus the Rg-0 and Rg-1 variants represent a form of "deep rough" mutation in M. avium. Separately, we report that these rough variants of M. avium differ genetically from the smooth, virulent form by major deletions of portions of the genes responsible for glycopeptidolipid synthesis (Belisle, J. T., Klaczkiewicz, K., Brennan, P. J., Jacobs, W. R., Jr., and Inamine, J. M. (1993) J. Biol. Chem. 268, 10517-10523). The isolation of different sets of spontaneous mutants of M. avium differentially defective in the capacity to synthesize glycopeptidolipids provides the means to explore their biosynthesis and roles in opportunistic pathogenesis.
Highlights
Gaschromatography,fastatombombardment-mass transparent (SmT) and avirulent smoothdomed (SmD) colspectrometry and‘H NMR to lipopeptideI established ony forms express GPLs, whereas those rough (Rg) variants the structureC,:,&OH-fatty acyl-D-Phe-D-allo-Thr- isolated to datehave variously been described as virulent [13]
A single 6-deoxytalose unit is attached to thDe-allo-Thr in the case of both the nsGPL and ssGPL
Deoxytalose is further glycosylated to yield a haptenic oligosaccharide that varies in composition among different sero
Summary
Were scraped, placed in sterile PBS with 0.05% Tween and stirred overnight to generate homogeneoussuspensions which werestored as Isolation of Morphological Variants of M. auium-Initial stocks at -70"C. TheSilica Gel was scraped from the plates, the lipids plating of the original SmD variant were picked and subculwere recovered by extraction with CHC13-CH30H(2:1), washed by partitioning with water, redissolved in CHCl,-CH,OH (9:1), and passed through an Acrodisc CR, 0.2-+mfilter (Gelman Sciences, Ann Arbor, MI) to remove remaining Silica Gel. The ssGPL of smooth variants of M. auiumserovar 2 was obtained as described [22]. They demonstrated pure morphology after the third passage, and, like Rg-0, these new rough isolates did not react with the anti-ssGPL-2 monoclonal antibody They were hydrolyzed with 2 M CF3COOH andthe resulting free Identification of a New Lipopeptide in Some Rg Variantssugars converted to alditol acetates and resolved on a DB 23 fused silica capillary column (J&W Scientific, Folsom,CA) as described [23]. Structures of Lipopeptides I and 11-To confirm the sequence of the amino acids within the peptidecore as well as the length and degree of unsaturation of the fatty acid, the
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