Abstract
The unique terminal arabinan motifs of mycobacterial lipoarabinomannan (LAM), which are mannose-capped to different extents, probably constitute the single most important structural entity engaged in receptor binding and subsequent immunopathogenesis. We have developed a concerted approach of endoarabinanase digestion coupled with chromatography and mass spectrometry analysis to rapidly identify and quantitatively map the complement of such terminal units among the clinical isolates of different virulence and drug resistance profiles. In comparison with LAM from laboratory strains of Mycobacterium tuberculosis, an ethambutol (Emb) resistant clinical isolate was shown to have a significantly higher proportion of nonmannose capped arabinan termini. More drastically, the mannose capping was completely inhibited when an Emb-susceptible strain was grown in the presence of subminimal inhibitory concentration of Emb. Both cases resulted in an increase of arabinose to mannose ratio in the overall glycosyl composition of LAM. Emb, therefore, not only could affect the complete elaboration of the arabinan as found previously for LAM from Mycobacterium smegmatis resistant mutant but also could inhibit the extent of mannose capping and hence its associated biological functions in M. tuberculosis. Unexpectedly, an intrinsically Emb-resistant Mycobacterium avium isolate of smooth transparent colony morphology was found to have most of its arabinan termini capped with a single mannose residue instead of the more common dimannoside as established for LAM from M. tuberculosis. This is the first report on the LAM structure from M. avium complex, an increasingly important opportunistic infectious agent afflicting AIDS patients.
Highlights
One of the most prominent macromolecular entities of all mycobacterial cell walls is the arabinan, a common constituent of both the arabinogalactan (AG)1 and lipoarabinomannan (LAM) [1]
We demonstrated in this paper that endoarabinanase digestion of intact LAM followed by direct high pH anion exchange chromatography (HPAEC) and mass spectrometry (MS) analysis can be a quick and effective way to assess the branching nature and capping functions of the arabinan motifs
We provided the first pictures of the native construct of the arabinan terminal motifs in LAMs from clinical isolates of M. tuberculosis and M. avium complex and highlighted their substantial differences in the nature of mannose capping
Summary
Ture and function definition because of its size, extreme heterogeneity, and the chemical nature of the arabinan. Given the particular biological relevance of the terminal arabinan structures, a rapid and high sensitivity method of defining their relative profiles is imperative to further understanding of the consequence of drug action and resistance. Toward this end, our own crude endoarabinanase preparation [16] provides the only presently available tool to release intact the variably capped terminal oligoarabinosyl motifs from LAM [5, 17] without resorting to chemical cleavage methods that would otherwise degrade the labile arabinofuranosyl bonds. For the first time, that mannose capping on LAM from the laboratory M. tuberculosis strain can be completely abolished when grown in the presence of Emb at a concentration that would be ineffective against the drug resistant clinical isolates
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