TRUNCATED STRUCTURAL VARIANTS OF LIPOARABINOMANNAN IN ETHAMBUTOL DRUG-RESISTANT STRAINS OF MYCOBACTERIUM-SMEGMATIS - INHIBITION OF ARABINANBIOSYNTHESIS BY ETHAMBUTOL

The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of arabinans of both the cell wall arabinogalact an (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by cons ecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a norm al version of AG, produced truncated forms of LAM when maintained on 1 0 mu g/ml Emb (Mikusova, K., Slayden, R. A., Besra, G. S., and Brennan , P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have n ow isolated and characterized the truncated LAMs made by both the resi stant mutant and a recombinant strain transfected with a plasmid conta ining the emb region from Mycobacterium avium which encodes for Emb re sistance. By chemical analysis, endoarabinanase digestion, high pH ani on exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, whi ch constitutes a substantial portion of the arabinan termini in LAM bu t not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulti ng in severely truncated LAM as well as AG with a reduced Ara:Gal rati o. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosy nthesis of arabinans unique to the mycobacterial cell wall. It was fur ther concluded that the uniquely branched terminal Ara(6) motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsi ble for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.