Disruption of Mycobacterial AftB Results in Complete Loss of Terminal β(1 → 2) Arabinofuranose Residues of Lipoarabinomannan

Lipoarabinomannan (LAM) and arabinogalactan (AG) are the two major mycobacterial cell wall (lipo)­polysaccharides, which contain a structurally similar arabinan domain that is highly branched and assembled in a stepwise fashion by variety of arabinofuranosyltransferases (Ara<i>f</i>T). In addition to playing an essential role in mycobacterial physiology, LAM and its biochemical precursor lipomannan possess potent immunomodulatory activities that affect the host immune response. In the search of additional mycobacterial Ara<i>f</i>Ts that participate in the synthesis of the arabinan segment of LAM, we disrupted <i>aftB</i> (<i>MSMEG_6400</i>) in <i>Mycobacterium smegmatis</i>. The deletion of chromosomal <i>aftB</i> locus could only be achieved in the presence of a rescue plasmid carrying a functional copy of <i>aftB</i>, strongly suggesting that it is essential for the viability of <i>M. smegmatis</i>. Isolation and detailed structural characterization of a LAM molecule derived from the conditional mutant deficient in AftB revealed the absence of terminal β(1 → 2)-linked arabinofuranosyl residues. Furthermore, we demonstrated that truncated LAM displays proinflammatory activity, which is due to its ability to activate Toll-like receptor 2. All together, our results indicate that AftB is an essential mycobacterial Ara<i>f</i>T that plays a role in the synthesis of the arabinan domain of LAM

Biosynthesis of the Methylthioxylose Capping Motif of Lipoarabinomannan in <i>Mycobacterium tuberculosis</i>

Lipoarabinomannan (LAM) is a lipoglycan found in abundant quantities in the cell envelope of all mycobacteria. The nonreducing arabinan termini of LAM display species-specific structural microheterogeneity that impacts the biological activity of the entire molecule. <i>Mycobacterium tuberculosis</i>, for instance, produces mannoside caps made of one to three α-(1 → 2)-Man<i>p</i>-linked residues that may be further substituted with an α-(1 → 4)-linked methylthio-d-xylose (MTX) residue. While the biological functions and catalytic steps leading to the formation of the mannoside caps of <i>M. tuberculosis</i> LAM have been well established, the biosynthetic origin and biological relevance of the MTX motif remain elusive. We here report on the discovery of a five-gene cluster dedicated to the biosynthesis of the MTX capping motif of <i>M. tuberculosis</i> LAM, and on the functional characterization of two glycosyltransferases, MtxS and MtxT, responsible, respectively, for the production of decaprenyl-phospho-MTX (DP-MTX) and the transfer of MTX from DP-MTX to the mannoside caps of LAM. Collectively, our NMR spectroscopic and mass spectrometric analyses of <i>mtxS</i> and <i>mtxT</i> overexpressors and knockout mutants support a biosynthetic model wherein the conversion of 5′-methylthioadenosine, which is a ubiquitous byproduct of spermidine biosynthesis, into 5′-methylthioribose-1-phosphate precedes the formation of a 5′-methylthioribose nucleotide sugar, followed by the epimerization at C-3 of the ribose residue, and the transfer of MTX from the nucleotide sugar to decaprenyl-phosphate yielding the substrate for transfer onto LAM. The conservation of the MTX biosynthetic genes in a number of Actinomycetes suggests that this discrete glycosyl substituent may be more widespread in prokaryotes than originally thought