The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3lketoacyl reductase that fails to confer isoniazid resistance

Banerjee, Asesh; Sugantino, Michele; Sacchettini, James C.; Jacobs, William R.

doi:10.1099/00221287-144-10-2697

Cited by 76 publications

(70 citation statements)

References 32 publications

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“…However, because such substrates are not commercially available, the assay relies on the use of acetoacetyl-CoA, although it has a poor affinity for the enzyme (19,20). Because KAR activity can be assessed by monitoring conversion of NADPH to NADP ϩ spectro photometrically, we examined if replacement of the Thr 191 primary phosphoacceptor by either Ala or Asp might affect binding to the NADPH cofactor.…”

Section: Kar Activity Is Reduced In Maba_t191a and Maba_t191d Mutantsmentioning

confidence: 99%

“…These are used as primers by the FAS-II system and iteratively condensed with malonyl-ACP in a reaction catalyzed by mtFabH, the ␤-ketoacyl-ACP synthase III of M. tuberculosis (16 -18). During the second step of the elongation cycle, the resulting ␤-ketoacyl-ACP product is reduced by MabA, the NADPH-dependent ␤-ketoacyl reductase of M. tuberculosis (19,20). The resulting ␤-hydroxyacyl-ACP is then dehydrated by a set of dehydratases, HadABC (21,22), and finally reduced by the enoyl-ACP reductase, InhA, the primary target of isoniazid (23).…”

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confidence: 99%

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Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Veyron‐Churlet

Zanella-Cléon

Cohen‐Gonsaud

et al. 2010

Journal of Biological Chemistry

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Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential ␤-ketoacylacyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr 191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr 191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

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Section: Kar Activity Is Reduced In Maba_t191a and Maba_t191d Mutantsmentioning

confidence: 99%

mentioning

confidence: 99%

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Veyron‐Churlet

Zanella-Cléon

Cohen‐Gonsaud

et al. 2010

Journal of Biological Chemistry

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“…For example, 96% to 100% of RIFresistant M. tuberculosis isolates have at least 1 mutation in rpoB, which encodes the RNA polymerase ␤-subunit (17, 31, 39). Of INH-resistant isolates, 42% to 58% have at least 1 mutation in katG, which encodes catalase-peroxidase, and 21% to 34% carry at least 1 mutation in the promoter of mabA, a synonym for fabG1 (10), which encodes a 3-ketoacyl reductase (3,17,38,39). Of EMB-resistant isolates, 47% to 65% have at least one mutation in embB, which encodes arabinosyltransferase (17,32,39).…”

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confidence: 99%

Detection of Multidrug Resistance in Mycobacterium tuberculosis

et al. 2007

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We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.The emergence and spread of drug-resistant strains of Mycobacterium tuberculosis, especially multidrug-resistant (MDR) strains, are serious threats to the control of tuberculosis and comprise an increasing public health problem (40). Patients infected with MDR strains, which are defined as strains resistant to both rifampin (RIF) and isoniazid (INH), are difficult to cure and are more likely to remain sources of infection for a longer period of time than are patients with drug-susceptible strains (40).It is essential that rapid drug susceptibility tests be developed to prevent the spread of MDR M. tuberculosis. The time necessary for culture of specimens was reduced by the radiometric BACTEC 460TB system (BD Biosciences, Sparks, MD), the nonradiometric ESP II system (Trek Diagnostics, Westlake, OH), and other rapid broth methods, such as BACTEC MGIT 960 SIRE (BD Biosciences) (20). These drug susceptibility tests, however, still require 1 to 2 weeks for final determination and reporting to the clinician (23). Additional reductions in the detection period are needed.Drug resistance in M. tuberculosis is caused by mutations in relatively restricted regions of the genome (17, 39). Mutations associated with drug resistance occur in rpoB for RIF, katG and the promote...

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“…Le mutazioni a carico del codone 315 del gene katG rappresentano il 60-80% dei casi INH-resistenti (INH-R). Sono state identificate mutazioni anche nelle regioni regolatorie di inhA e ahpC-oxyR (3,9,11). L'analisi di ceppi di MTB INH-R isolati in diver-si Paesi ha dimostrato che la prevalenza di tali mutazioni varia su scala geografica (16).…”

Section: Introduzioneunclassified

Identificazione molecolare di mutazioni conferenti resistenza a rifampicina ed isoniazide in M. tuberculosis in campioni clinici diretti mediante Genotype MTBDR (Hain Lifescience)

Miotto¹,

Piana²,

Penati³

et al. 2007

Microbiol Med

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Identification of mutations conferring rifampin and isonizid resistance in clinical samples using the Genotype MTBDR (Hain Lifescience) SUMMARYThe widespread of multi-drug resistant (MDR) Mycobacterium tuberculosis, resistant at least to rifampin (RIF) and isoniazid (INH), represents serious threats to the control of tuberculosis (TB) and increases the public health challenge worldwide. Surveillance program and rapid identification of drug-resistance strains are key-elements for an early and appropriate TB management. The aim is to evaluate the Genotype MTBDR (Hain Lifescience, Nehren, Germany), a reverse hybridization-based assay, as a rapid tool to identify mutations in the rpoB and katG genes associated to RIF-/INH-resistance directly in clinical specimens.We also evaluate the performance of a paper based device (Genocard -Hain Lifescience, Nehren, Germany) to collect and transport inactivated biological material. The test was evaluated retrospectively on 68 respiratory samples with positive cultures for M. tuberculosis. Considering the smear-positive samples only, the Genotype MTBDR gave interpretable results in 56 out of 57 samples (98.2%). The main limitations of the Genotype MTBDR are the difficulties in the amplification from smear-negative samples and the low sensitivity for the INH-resistance. The inclusion of probes targeting other regions involved in INH-resistance will increase the sensitivity of the test. The GenoCard, with its easy-and rapid-to use features, represents a functional tool for the sample collection with cost-effective and bio-safety benefits.The possibility to use the GenoCard directly in amplification reactions facilitates the gathering of data by molecular approaches.

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The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3lketoacyl reductase that fails to confer isoniazid resistance

Abstract: A target of the anti-tuberculosis drugs isoniazid (INH) and ethionamide (ETH

Cited by 76 publications

References 32 publications

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

Detection of Multidrug Resistance in Mycobacterium tuberculosis

Identificazione molecolare di mutazioni conferenti resistenza a rifampicina ed isoniazide in M. tuberculosis in campioni clinici diretti mediante Genotype MTBDR (Hain Lifescience)

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