Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the ...so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In
Mycobacterium tuberculosis isoniazid (INH)-susceptibility and the presence of a thermolabile catalase-peroxidase (T-catalase) are nearly always associated. It is shown in this study that an ...INH-susceptible strain of
M. aurum had a T-catalase activity while its resistant mutants did not, but an in vitro susceptible strain of
M. avium had a strong catalase activity without any detectable peroxidase properties. Synthesis of mycolic acids is a genus-specific target for INH and there is an excellent parallelism between INH-susceptibility of intact cells and that of a cell-free system synthesizing mycolic acids. We investigated whether the INH-inhibition of mycolic acid cell-free synthesis was dependent on a T-catalase activity in
M. aurum and
M. avium: no catalase activity was detectable in any of the cell-free systems tested, and addition of T-catalase from susceptible
M. aurum strain to an INH-resistant system did not render it sensitive. So INH can inhibit mycolic acid synthesis independently of the presence of a T-catalase. An INH-susceptible cell-free system prepared from INH-treated (at the MIC) cells was progressively and irreversibly inhibited, while incubation of the same susceptible system in the presence of INH did not result in a significant irreversible inhibition. The possible participation of T-catalase in the irreversible effect of INH is discussed.
The hypothetical schemes proposed for the biosynthesis of unsaturated mycolic acids (R1-CH(OH)-CH(R2)-COOH) of Mycobacteria cell walls were experimentally tested by using cell-free extracts either of ...Mycobacterium aurum or of Mycobacterium smegmatis which produce two kinds of unsaturated mycolic acids (mono and dialkene), 1-14Cacetate being the precursor. Examination of specific radioactivities, in the presence or in the absence of isoniazid, an antituberculous drug inhibiting mycolic acid synthesis, showed that saturated C22 and C24 acids play a role as precursors of two distinct parts of the mycolic acids. Moreover, determination of labelling distribution into mycolic acid fragments obtained by oxidative and pyrolytic cleavages showed first that the side chain R2 and the methyl end R1 both have these C22 and C24 saturated fatty acids as common precursors. Secondly, it is thought that the fragments located between the methyl end R1 and the side chain R2 mainly result from elongation steps (one or two successive additions of seven or eight C2 units according to the mycolic acid type) and a biosynthetic model is proposed for unsaturated mycolic acids extending the published models and illustrating the missing step in monoalkene formation.
Resistance to isoniazid in Mycobacterium tuberculosis can be mediated by substitution of alanine for serine 94 in the InhA protein, the drug's primary target. InhA was shown to catalyze the ...beta-nicotinamide adenine dinucleotide (NADH)--specific reduction of 2-transenoyl-acyl carrier protein, an essential step in fatty acid elongation. Kinetic analyses suggested that isoniazid resistance is due to a decreased affinity of the mutant protein for NADH. The three-dimensional structures of wild-type and mutant inhA, refined to 2.2 and 2.7 angstroms, respectively, revealed that drug resistance is directly related to a perturbation in the hydrogen-bonding network that stabilizes NADH binding.
The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III)-pyrophosphate. In the presence of the nicotinamide coenzyme, the INH oxidation produced the ...formation of INH-NAD(H) adducts and allowed the in vitro inhibition of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway for mycolic acids. Manganese(III)-pyrophosphate is an efficient alternative oxidant to mimic the activity of the
Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations on reactive INH species and resulting InhA inhibitors.
Version française abrégée – Inhibition de l’énoyl-
Acyl Carrier Protein
réductase InhA de
Mycobacterium tuberculosis
par l'isoniazide activé par le pyrophosphate de manganèse(III) en présence de NADH. L'isoniazide (INH) est un antibiotique antituberculeux qui, après une étape de bioactivation oxydante par la catalase-peroxydase KatG de
Mycobacterium tuberculosis en présence du coenzyme NAD(H), est capable d'inhiber l’énoyl-
Acyl Carrier Protein réductase InhA, l'une des cibles de l'INH dans la voie de biosynthèse des acides mycoliques, constituants spécifiques de la paroi bactérienne. La formation d'adduits covalents INH–NAD(H) comme inhibiteurs de InhA est une hypothèse couramment évoquée pour expliquer cet effet. Dans ce travail, nous décrivons la possibilité d'utiliser le pyrophosphate de manganèse(III) comme un réactif chimique utilisable à la place de la protéine KatG, non aisément accessible, ou du système manganèse(II)/O
2, qui nécessite des périodes d'incubation de plusieurs heures. Nous avons montré que le pyrophosphate de manganèse(III) est effectivement capable, lorsqu'il est utilisé en quantité stœchiométrique, d'oxyder INH très efficacement (conversion supérieure à 90
%,
tableau
I
, entrée
1) et rapidement (en moins de 15
min) avec formation d'acide isonicotinique, d'isonicotinamide et d'isonicotinaldéhyde (
schéma1
), trois produits qui sont ceux formés lors de l'oxydation d'INH catalysée par la protéine KatG
3. Lorsque cette même oxydation est conduite en présence des coenzymes NAD
+ ou NADH, l'analyse par CLHP permet de montrer la formation d'une famille d'adduits présentant des caractéristiques d'absorption UV (
λ
max à 230 et à 320–333
nm) et de spectrométrie de masse (masse moléculaire 770,1) attribuables à une structure de type dihydropyridine, comme celles récemment décrites par Johnsson et al.
7. En présence du coenzyme NAD(H) et de l’énoyl-
Acyl Carrier Protein réductase InhA, l'oxydation d'INH par le pyrophosphate de manganèse(III) conduit à l'inhibition rapide et efficace d'InhA (79
% de perte d'activité d'InhA après 12
min d'incubation). Aussi ce système chimique d'activation d'INH doit pouvoir très utilement remplacer la catalase-peroxydase KatG présente chez
Mycobacterium tuberculosis, notamment pour des études mécanistiques sur le mode d'action d'INH, ainsi que pour élucider les structures des espèces activées mises en jeu et celles des inhibiteurs qui en résultent.
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