Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria. © 2001 Federation of European Microbiological Societies.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Frequency of Mycobacterium chimaera among Belgian patients, 2015

Mycobacterium chimaera arouses an increasing public health concern, as this non-tuberculous mycobacteria (NTM) has recently been associated with life-threatening cardiac infections. M. chimaera and M. intracellulare are genetically very close, but recently appeared to present different epidemiological and clinical significance. Therefore, it has become important for laboratories to use adequate techniques allowing a precise species identification. To date, most commercially available laboratory assays cannot distinguish them, and erroneously identify M. chimaera as M. intracellulare. We performed a re-analysis of the 149 M. intracellulare strains received by the Belgian National Reference Laboratory using 16S rRNA gene sequencing, representing 25 % of all NTM collected in 2015. We found that M. chimaera represents the majority (n=94, 63 %) of the previously M. intracellulare. This study reports the large presence of M. intracellulare/chimaera among Belgian patients infected by a NTM and the predominance of the species M. chimaera among this group. This study also stresses the public health importance of M. chimaera and demonstrates the inability of commonly-used laboratory techniques to correctly diagnose these infections

Erratum - Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C (FEMS Microbiology Letter (2001) 196 (51-56) PII: S0378109701000350)

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Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase, a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis.

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.info:eu-repo/semantics/publishe

Telacebec Interferes with Virulence Lipid Biosynthesis Protein Expression and Sensitizes to Other Antibiotics

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a public health issue, particularly due to multi-drug-resistant Mtb. The bacillus is wrapped in a waxy envelope containing lipids acting as essential virulence factors, accounting for the natural antibiotic resistance of mycobacteria. Telacebec (previously known as Q203) is a promising new anti-TB agent inhibiting the cytochrome bc1 complex of a mycobacterial electron transport chain (ETC). Here, we show that the telacebec-challenged M. bovis BCG exhibited a reduced expression of proteins involved in the synthesis of phthiocerol dimycocerosates (PDIMs)/phenolic glycolipids (PGLs), lipid virulence factors associated with cell envelope impermeability. Consistently, telacebec, at concentrations lower than its MIC, downregulated the transcription of a PDIM/PGL-synthesizing operon, suggesting a metabolic vulnerability triggered by the drug. The drug was able to synergize on BCG with rifampicin or vancomycin, the latter being a drug exerting a marginal effect on PDIM-bearing bacilli. Telacebec at a concentration higher than its MIC had no detectable effect on cell wall PDIMs, as shown by TLC analysis, a finding potentially explained by the retaining of previously synthesized PDIMs due to the inhibition of growth. The study extends the potential of telacebec, demonstrating an effect on mycobacterial virulence lipids, allowing for the development of new anti-TB strategies.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Isoniazid bactericidal activity involves electron transport chain perturbation

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Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Increased vancomycin susceptibility in mycobacteria: a new approach to identify synergistic activity against multi-drug resistant mycobacteria.

Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing M. bovis BCG and M. tuberculosis mutants, lacking phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids, with wild-type strains, we observed that glycopeptides strongly inhibited PDIM deprived mycobacteria. Vancomycin together with a drug targeting lipids synthesis inhibited multidrug-resistant (MDR) and extensively-drug resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential anti-TB agents and might provide a new antimycobacterial drug-screening strategy.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The active form of the <i>Mtb</i> MDH consists in a dimer.

<p>A: Native polyacrylamide gel (8%) was stained for MDH activity as described under Materials and Methods. B: Autoradiography of the gel. Lane 1: 2 μg of non-phosphorylated MDH; Lane 2: 2 μg of MDH phosphorylated by 0.25 μg of PknD; Lane 3: 0.25 μg of autophosphorylated PknD.</p