Search

The sensitivity of Mycobacterium tuberculosis , the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis , which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

The sensitivity of

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 mu g/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of C-14-uracil and C-14-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 mu g/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 mu g/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments., info:eu-repo/semantics/published

Countering TB prodrug resistance The arsenal of antibiotics for treating tuberculosis (TB) contains many prodrugs, such as ethionamide, which need activation by normal metabolism to release their toxic effects. Ethionamide is potentiated by small molecules. Blondiaux et al. screened for more potent analogs and identified a lead compound called SMARt-420. This small molecule inactivates a TetR-like repressor, EthR2, and boosts ethionamide activation. SMARt-420 successfully promoted clearance of multidrug-resistant strains of Mycobacterium tuberculosis from the lungs of mice. Science , this issue p. 1206

Tuberculosis is still a cause of major concern, partly due to the emergence of multidrug-resistant strains. New drugs are therefore needed. Vancomycin can target mycobacteria with cell envelope deficiency. In this study, we used a vancomycin susceptibility assay to detect drugs hampering lipid synthesis in Mycobacterium bovis BCG and in Mycobacterium tuberculosis . We tested three drugs already used to treat human obesity: tetrahydrolipstatin (THL), simvastatin, and fenofibrate. Only vancomycin and THL were able to synergize on M. bovis BCG and on M. tuberculosis , although mycobacteria could also be inhibited by simvastatin alone. Lipid analysis allowed us to identify several lipid modifications in M. tuberculosis H37Rv treated with those drugs. THL treatment mainly reduced the phthiocerol dimycocerosate (PDIM) content in the mycobacterial cell wall, providing an explanation for the synergy, since PDIM deficiency has been related to vancomycin susceptibility. Proteomic analysis suggested that bacteria treated with THL, in contrast to bacteria treated with simvastatin, tried to recover, inducing, among other reactions, lipid synthesis. The combination of THL and vancomycin should be considered a promising solution in developing new strategies to treat multidrug-resistant tuberculosis.

The transcriptional regulator EthR from Mycobacterium tuberculosis, a member of the TetR family of prokaryotic homo-dimeric transcriptions factors, controls the expression of the mycobacterial mono-oxygenase EthA. Due to the fact that EthA is responsible for the bio-activation of the second-line tuberculosis pro-drug ethionamide, EthR inhibitors have been shown to boost drug efficacy by increasing EthA levels. Here, we present a comprehensive in-silico structure-based screening protocol that led to the identification of a number of novel scaffolds of EthR inhibitors. We present biophysical characterization of 85 potential leads, 20 of which showed binding by thermal shift assays. The co-crystal structures of EthR with four new ligands at resolution ranging from 2.1 to 1.4 Å confirm the binding and inactivation mode. The crystal structures include ligands with three new chemical scaffolds that will enable future lead development. Five of the lead compounds showed the desired booster effect with the most promising displaying an EC50 value of 0.76 μM.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant

Deletion of genes in a pathogen is commonly associated with a reduction in its ability to cause disease. However, some rare cases have been described in the literature whereby deletion of a gene results in an increase in virulence. Recently, there have been several reports of hypervirulence resulting from gene deletion in Mycobacterium tuberculosis. Here, we explore this phenomenon in the context of the interaction between the pathogen and the host response.

The Mycobacterium tuberculosis TetR-type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome-wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real-time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system.

Bacterial pathogens adapt to their host environments to a large extent through switching on complex transcriptional programmes, and whole-genome microarray experiments promise to reveal this complexity. There has been a recent burst of articles reporting transcriptome analyses of Mycobacterium tuberculosis, including for the first time studies in macrophages and mice. We review gene expression reports, and compare them with each other and with microarray-based gene essentiality studies, revealing at times a startling lack of correlation. Additionally, we suggest a standardization format for the submission of processed data for publication, to facilitate cross-experiment analyses.

In malaria, hemozoin (Hz) is released during erythrocyte rupture, and subsequent phagocytosis appears to cause important immune-modulatory effects. Hz obtained from Plasmodium falciparum cultures or synthesized from heme is used to study this modulation in vitro. Immune-activating and suppressive effects have been reported, and these discrepant results are often attributed to the different types of Hz that were used. However, it remains unclear which type of Hz accurately reproduces what happens in vivo. Importantly, Hz remains in the body for long periods and appears to be actively redistributed. Thus, phagocytosis of Hz in the body is not static but probably happens more than once, and the characteristics of Hz may change over time, eventually causing different immune-modulatory effects.

The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level.

The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cassette. A selection of marker genes obtained from the pGOAL series of vectors are inserted into the pNIL vector to create a suicide delivery system. This delivery vector is introduced into mycobacteria where the disrupted version of the gene replaces the wild-type version by a two-step homologous recombination process. The method involves selecting for a single crossover event followed by selection of double crossovers. Single crossovers have incorporated plasmid marker genes and are sucrose(S), kanamycin(R) and blue on media containing X-gal. Double crossovers have lost plasmid markers and are sucrose(R), kanamycin(S) and white on media containing X-gal.

Background Detection of malaria pigment (or haemozoin; Hz)-containing leukocytes may have prognostic relevance in malaria; however, studies reported conflicting results, with microscopic counts suggestive of being inaccurate and imprecise. Methods Numbers of Hz-containing leukocytes from a malaria patient obtained with a flow cytometer counting 50.000 gated events were compared with thin film microscopy as applied under field conditions. Results Flow cytometry identified 5.8% Hz-containing monocytes and 1.8% Hz-containing neutrophils. The microscopic examination yielded 10% and 13% of Hz-containing monocytes, as well as 0% and 0.5% of Hz-containing neutrophils for observers one and two, respectively. Conclusion Novel, robust and affordable cytometric methods should be evaluated in the field as they may assist in utilizing Hz-containing cells as clinically useful parameter.

The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cassette. A selection of marker genes obtained from the pGOAL series of vectors are inserted into the pNIL vector to create a suicide delivery system. This delivery vector is introduced into mycobacteria where the disrupted version of the gene replaces the wild-type version by a two-step homologous recombination process. The method involves selecting for a single crossover event followed by selection of double crossovers. Single crossovers have incorporated plasmid marker genes and are sucrose(S), kanamycin(R) and blue on media containing X-gal. Double crossovers have lost plasmid markers and are sucrose(R), kanamycin(S) and white on media containing X-gal.