An integrated computational investigation to unveil the structural impacts of mutation on the InhA structural gene of Mycobacterium tuberculosis

ABSTRACT Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were ≥50 μg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were ≥100 μg/ml, and these isolates were also resistant to ≥8 μg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were ≥32 μg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to ≥50 μg of ETH per ml had such mutations.

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Computational investigation of phytomolecules as resuscitation-promoting factor B (RpfB) inhibitors for clinical suppression of Mycobacterium tuberculosis dormancy reactivation

Infection Genetics and Evolution ◽

10.1016/j.meegid.2020.104356 ◽

2020 ◽

Vol 83 ◽

pp. 104356

Author(s):

Vivek Dhar Dwivedi ◽

Aditya Arya ◽

Tanuj Sharma ◽

Shweta Sharma ◽

Shripad A. Patil ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Computational Investigation ◽

Factor B

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Screening and Characterization of Mutations in Isoniazid-Resistant Mycobacterium tuberculosis Isolates Obtained in Brazil

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3373-3381.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3373-3381 ◽

Cited By ~ 60

Author(s):

Rosilene Fressatti Cardoso ◽

Robert C. Cooksey ◽

Glenn P. Morlock ◽

Patricia Barco ◽

Leticia Cecon ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Dna Sequencing ◽

Structural Gene ◽

Intergenic Region ◽

Promoter Region ◽

Regulatory Region ◽

Gene Mutations ◽

Novel Mutations ◽

Single Strand Conformational Polymorphism

ABSTRACT We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide −48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G269A substitution. Although KatG 315 substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

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The MTCY428.08 Gene ofMycobacterium tuberculosis Codes for NAD+ Synthetase

Journal of Bacteriology ◽

10.1128/jb.180.12.3218-3221.1998 ◽

1998 ◽

Vol 180 (12) ◽

pp. 3218-3221 ◽

Cited By ~ 16

Author(s):

Rita Cantoni ◽

Manuela Branzoni ◽

Monica Labò ◽

Menico Rizzi ◽

Giovanna Riccardi

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Structural Gene ◽

Sequence Homology ◽

Expression Vectors ◽

Synthetase Activity ◽

Gene Products ◽

Ofmycobacterium Tuberculosis ◽

Nad Synthetase

ABSTRACT The product of the MTCY428.08 gene ofMycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase.

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