New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual ␤-(134) and ␤-(135) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.The mycobacterial cell wall-including the essential, covalently linked complex of peptidoglycan, heteropolymeric arabinogalactan, and highly hydrophobic mycolic acids-is responsible for many of the pathophysiological features of members of the Mycobacterium genus (9). Several antituberculosis drugs affect the formation of this complex. Isoniazid, ethionamide, thiocarlide, and thiacetazone inhibit mycolic acid synthesis (14,29,30,36,38); ethambutol specifically disrupts the synthesis of arabinan (20,24,35,39); and D-cycloserine, an inhibitor of peptidoglycan synthesis (11), has some clinical utility. However, drug resistance, particularly the multiple and extensive forms, is of pressing public health concern (15, 31), presaging the need for a broader array of targets and drugs affecting both cell wall synthesis and other aspects of mycobacterial metabolism. However, our understanding of the synthesis of the mycobacterial cell wall is elementary compared to that of other bacteria.The initiation point for arabinogalactan biogenesis is the mycobacterial version of the bacterial carrier lipid, bactoprenol, identified as decaprenyl phosphate (C 50 -P) (10), and the sequential addition of GlcNAc-P, rhamnose (Rha), and single galactofuranose (Galf) units, donated by the appropriate nucleotide sugars (23, 25) (Fig. 1). At some stage, the arabinofuranose (Araf) units are added, donated not by a nucleotide sugar but a lipid carrier, C 50 -P-Araf (20, 39). Several of the responsible glycosyl transferases taking part in this process have been identified (1,3,5,18,19,25,26,32,34).We recently described the galactofuranosyl transferase, Rv3782, responsible for attaching the first and, perhaps, the second Galf unit to the C 50 -P-P-GlcNAc-Rha acceptor (22). Due to its role in the initiation of galactan formation, we now name it galactofuranosyl transferase 1 (GlfT1). Previously, yet another galactofuranosyl transferase, Rv3808c (originally called GlfT but now named GlfT2), was recognized and proved to be bifunctional in that it was responsible for the formation of the bulk of the galactan, containing alternating 5-and 6-linked ␤-Galf units (19,25,32). In the present study, we examine the precise roles of GlfT1 and GlfT2 in mycobacterial cell wall galactan synthesis through the application of in vitro reactions with purified natural acc...

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

The arabinogalactan (AG) of slow growing pathogenic Mycobacterium spp. is characterized by the presence of galactosamine (GalN) modifying some of the interior branched arabinosyl residues. The biosynthetic origin of this substituent and its role(s) in the physiology and/or pathogenicity of mycobacteria are not known. We report on the discovery of a polyprenylphospho-N-acetylgalactosaminyl synthase (PpgS) and the glycosyltransferase Rv3779 from Mycobacterium tuberculosis required, respectively, for providing and transferring the GalN substrate for the modification of AG. Disruption of either ppgS (Rv3631) or Rv3779 totally abolished the synthesis of the GalN substituent of AG in M. tuberculosis H37Rv. Conversely, expression of ppgS in Mycobacterium smegmatis conferred upon this species otherwise devoid of ppgS ortholog and any detectable polyprenyl-phospho-N-acetylgalactosaminyl synthase activity the ability to synthesize polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc. Interestingly, this catalytic activity was increased 40 -50-fold by co-expressing Rv3632, the encoding gene of a small membrane protein apparently co-transcribed with ppgS in M. tuberculosis H37Rv. The discovery of this novel lipid-linked sugar donor and the involvement of a the glycosyltransferase C-type glycosyltransferase in its transfer onto its final acceptor suggest that pathogenic mycobacteria modify AG on the periplasmic side of the plasma membrane.The availability of a ppgS knock-out mutant of M. tuberculosis provides unique opportunities to investigate the physiological function of the GalN substituent and the potential impact it may have on host-pathogen interactions.Galactosamine (D-GalN) was recognized as a minor covalently bound amino sugar component of the cell wall of slow growing mycobacteria (Mycobacterium tuberculosis H37Ra, Mycobacterium lepraemurium, Mycobacterium microti) as early as in the 1970s (1-3). Advances in mass spectrometry later coupled with the use of an arabinanase isolated from Mycobacterium smegmatis allowed the GalN substituent to be mapped to the arabinogalactan (AG) 7 component of the cell wall (3), and more specifically to the C2 position of a portion of the internal 3,5-branched D-Araf residues in the AG of M. tuberculosis (4, 5). D-GalN was estimated to occur at the level of about one residue per entire AG molecule (3-5). Interestingly, a similar sugar residue was found to substitute the AG of Mycobacterium avium, Mycobacterium kansasii, Mycobacterium bovis BCG (3) and Mycobacterium leprae, 8 but not that of M. smegmatis, M. neoaurum, and M. phlei (3-5), suggesting that fast growing Mycobacterium spp. are devoid of GalN substituent.The function and biosynthetic origin of this substituent are not known. Non-N-acetylated hexosamine residues, particularly GalN residues, are rather uncommon in prokaryotes. Thus far, they essentially have been described as components of the lipopolysaccharide (LPS) of some Gram-negative bacteria (6 -9). In Francisella tularensis a...

SUMMARY UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short “dead-end” intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.