An ethA-ethR-deficient Mycobacterium bovis BCG mutant displays increased adherence to mammalian cells and greater persistence in vivo, which correlate with altered mycolic acid composition.

Tuberculosis remains a major worldwide epidemic because of its sole etiological agent, Mycobacterium tuberculosis. Ethionamide (ETH) is one of the major antitubercular drugs used to treat infections with multidrug-resistant M. tuberculosis strains. ETH is a prodrug that requires activation within the mycobacterial cell; its bioactivation involves the ethA-ethR locus, which encodes the monooxygenase EthA, while EthR is a transcriptional regulator that binds to the intergenic promoter region of the ethA-ethR locus. While most studies have focused on the role of EthA-EthR in ETH bioactivation, its physiological role in mycobacteria has remained elusive, although a role in bacterial cell detoxification has been proposed. Moreover, the importance of EthA-EthR in vivo has never been reported on. Here we constructed and characterized an EthA-EthR-deficient mutant of Mycobacterium bovis BCG. Our results indicate that absence of the ethA-ethR locus led to greater persistence of M. bovis BCG in the mouse model of mycobacterial infection, which correlated with greater adherence to mammalian cells. Furthermore, analysis of cell wall lipid composition by thin-layer chromatography and mass spectrometry revealed differences between the ethA-ethR KO mutant and the parental strain in the relative amounts of α- and keto-mycolates. Therefore, we propose here that M. bovis BCG ethA-ethR is involved in the cell wall-bound mycolate profile, which impacts mycobacterial adherence properties and in vivo persistence. This study thus provides some experimental clues to the possible physiological role of ethA-ethR and proposes that this locus is a novel factor involved in the modulation of mycobacterial virulence.

Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB, and its role in benzothiazinone resistance.

Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-ß-d-ribose 2'-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.

Investigation of ABC transporter from mycobacterial arabinogalactan biosynthetic cluster.

Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc(2)155, phenotypic characterization of the mutant strains via metabolic labeling with [U-(14)C]-glucose, cell-free reactions with UDP-[U-(14)C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.

Galactosyl transferases in mycobacterial cell wall synthesis.

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 beta-(1-->4) and beta-(1-->5) 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.

A split-protein sensor for studying protein-protein interaction in mycobacteria.

The study of protein function in living cells is an essential complement to genomics, yet method development does not always keep pace with sequencing. Experimental techniques for the genus mycobacteria are relatively underdeveloped, though seventeen genomes have been sequenced. "Split-Trp" is a split-protein sensor used to detect protein-protein interactions in tryptophan auxotrophic Saccharomyces cerevisiae, but the principles behind the sensor should allow it to function in a broad range of microbial hosts. Here we introduce Split-Trp to Escherichia coli and Mycobacterium smegmatis and demonstrate that this system is a simple assay for protein interaction in both organisms.

Synthetic UDP-furanoses as potent inhibitors of mycobacterial galactan biogenesis.

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.

Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

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.