Isolation, characterization, and function of 6-mycolyl-6'-acetyltrehalose in the H37Ra strain of Mycobacterium tuberculosis

Abstract

The major mycolic acid containing extractable lipid of the H37Ra strain of Mycobacterium tuberculosis was established to be 6-mycolyl-6'-acetyltrehalose (MAT). This new glycolipid was extracted from harvested cells with chloroform-methanol (2:1, v/v) and initially precipitated out in acetone. A series of column (DEAE-cellulose, silicic acid, and Sephadex LH-20) and preparative thin-layer chromatography steps yielded a homogeneous preparation. A single sugar was released by saponification and it was identified to be trehalose by paper chromatography and gas-liquid chromatography of the trimethylsilyl derivative. The lipid moiety was determined to be exclusively mycolic acids. The major mycolic acid component of this glycolipid was isolated, purified as the methyl ester, and characterized to be methyl alpha-mycolate by nuclear magnetic resonance spectroscopy and mass spectrometry. The molar ratio of trehalose to mycolic acids was determined to be 1:1. The other acyl group in MAT was established to be acetate by gas-liquid chromatography. Methylation analysis showed the mycolate and acetate ester linkages to the 6 and 6' positions of trehalose. The time course of incorporation of 14C-labeled acetate into the mycolates of both MAT and total cellular fatty acids was followed. These results showed that the synthesis of MAT is rapid and linear for the initial 20 min of incubation whereas the curve for the total cellular mycolates minus MAT (an estimate of the cell wall mycolates) had a 25-30-min lag period. When the label in the lipids was chased out with an excess of unlabeled acetate, relatively rapid decline in the labeled MAT resulted with a corresponding rise in the level of radioactivity in the mycolates of the nonextractable cellular fraction (assumed to be the cell wall fraction). Thus mycolic acids are rapidly transferred from MAT to the cell wall of M. tuberculosis.