A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was P , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level.
View Article and Find Full Text PDFA handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the istone-like ucleoid-tructuring protein, is one of these NAPs and protects from foreign gene expression. Though lacking any sequence similarity with H-NS, Rv3852 was annotated as the H-NS ortholog in , as it resembles human histone H1.
View Article and Find Full Text PDFThere is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis.
View Article and Find Full Text PDFAntimicrob Agents Chemother
November 2016
Benzothiazinones (BTZs) are a class of compounds found to be extremely potent against both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains. The potency of BTZs is explained by their specificity for their target decaprenylphosphoryl-d-ribose oxidase (DprE1), in particular by covalent binding of the activated form of the compound to the critical cysteine 387 residue of the enzyme. To probe the role of C387, we used promiscuous site-directed mutagenesis to introduce other codons at this position into dprE1 of M.
View Article and Find Full Text PDFUnlabelled: Mycobacterium tuberculosis possesses a thick and highly hydrophobic cell wall principally composed of a mycolyl-arabinogalactan-peptidoglycan complex, which is critical for survival and virulence. DprE1 is a well-characterized component of decaprenyl-phospho-ribose epimerase, which produces decaprenyl-phospho-arabinose (DPA) for the biosynthesis of mycobacterial arabinans. Upstream of dprE1 lies rv3789, which encodes a short transmembrane protein of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides.
View Article and Find Full Text PDF8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity.
View Article and Find Full Text PDFThe flavo-enzyme DprE1 catalyzes a key epimerization step in the decaprenyl-phosphoryl d-arabinose (DPA) pathway, which is essential for mycobacterial cell wall biogenesis and targeted by several new tuberculosis drug candidates. Here, using differential radiolabeling with DPA precursors and high-resolution fluorescence microscopy, we disclose the unexpected extracytoplasmic localization of DprE1 and periplasmic synthesis of DPA. Collectively, this explains the vulnerability of DprE1 and the remarkable potency of the best inhibitors.
View Article and Find Full Text PDFPhenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against intracellular bacteria.
View Article and Find Full Text PDFThe cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M.
View Article and Find Full Text PDFImproved genetic tools are required to identify new drug targets in Mycobacterium tuberculosis. To this aim, genetic approaches, targeting either transcription and/or protein degradation, have been developed to appraise gene essentiality and to test the impact of gene silencing on bacterial survival. Here, we successfully combined the Tet-Pip OFF system, which downregulates transcription through the TetR and Pip repressors, with SspB-mediated protein degradation to study depletion of the transketolase encoded by the tkt (rv1449c) gene.
View Article and Find Full Text PDFThe complex multiprotein systems for the assembly of protein-bound iron-sulfur (Fe-S) clusters are well defined in Gram-negative model organisms. However, little is known about Fe-S cluster biogenesis in other bacterial species. The ISC (iron-sulfur cluster) operon of Mycobacterium tuberculosis lacks several genes known to be essential for the function of this system in other organisms.
View Article and Find Full Text PDFIn Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival.
View Article and Find Full Text PDFAs part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes.
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