Insights into the Mechanism of Ethionamide Resistance in Mycobacterium tuberculosis through an in silico Structural Evaluation of EthA and Mutants Identified in Clinical Isolates

Mutation in the ethionamide (ETH) activating enzyme, EthA, is the main factor determining resistance to this drug, used to treat TB patients infected with MDR and XDR Mycobacterium tuberculosis isolates. Many mutations in EthA of ETH resistant (ETH-R) isolates have been described but their roles in resistance remain uncharacterized, partly because structural studies on the enzyme are lacking. Thus, we took a two-tier approach to evaluate two mutations (Y50C and T453I) found in ETH-R clinical isolates. First, we used a combination of comparative modeling, molecular docking, and molecular dynamics to build an EthA model in complex with ETH that has hallmark features of structurally characterized homologs. Second, we used free energy computational calculations for the reliable prediction of relative free energies between the wild type and mutant enzymes. The ΔΔG values for Y50C and T453I mutant enzymes in complex with FADH2-NADP-ETH were 3.34 (+/−0.55) and 8.11 (+/−0.51) kcal/mol, respectively, compared to the wild type complex. The positive ΔΔG values indicate that the wild type complex is more stable than the mutants, with the T453I complex being the least stable. These are the first results shedding light on the molecular basis of ETH resistance, namely reduced complex stability of mutant EthA.

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Insights into the Mechanism of Ethionamide Resistance in Mycobacterium tuberculosis through an in silico Structural Evaluation of EthA and Mutants Identified in Clinical Isolates

10.20944/preprints202003.0470.v1 ◽

2020 ◽

Author(s):

Vinicius Carius de Souza ◽

Deborah Antunes ◽

Luciana H.S. Santos ◽

Priscila Vanessa Zabala Capriles Goliatt ◽

Ernesto Raul Caffarena ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Comparative Modeling ◽

Clinical Isolates ◽

Free Energies ◽

Wild Type ◽

Structural Evaluation ◽

Type Complex ◽

Computational Calculations ◽

First Results ◽

Delta G

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Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

Scientific Reports ◽

10.1038/s41598-020-80213-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ruiqi Wang ◽

Kun Li ◽

Jifang Yu ◽

Jiaoyu Deng ◽

Yaokai Chen

Keyword(s):

Mycobacterium Tuberculosis ◽

Type Strain ◽

Wild Type Strain ◽

Clinical Isolates ◽

Aminobenzoic Acid ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Expression Levels ◽

Encoding Gene ◽

Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

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pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates in Portugal

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.7.2736-2738.2004 ◽

2004 ◽

Vol 48 (7) ◽

pp. 2736-2738 ◽

Cited By ~ 28

Author(s):

Isabel Portugal ◽

Luís Barreiro ◽

José Moniz-Pereira ◽

Laura Brum

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Nucleotide Sequences ◽

Clinical Isolates ◽

Wild Type

ABSTRACT The nucleotide sequences of the pncA genes within 55 multidrug-resistant pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates were determined. Fifty-three out of the 55 isolates were pyrazinamidase (PZase) negative. Four strains contained a wild-type pncA gene, and PZase activity was undetectable in two of these strains. Seven of the 18 identified pncA mutations found have not been described in previous studies.

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Fluoroquinolone Action against Clinical Isolates ofMycobacterium tuberculosis: Effects of a C-8 Methoxyl Group on Survival in Liquid Media and in Human Macrophages

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.3.661 ◽

1999 ◽

Vol 43 (3) ◽

pp. 661-666 ◽

Cited By ~ 74

Author(s):

Ben Yang Zhao ◽

Richard Pine ◽

John Domagala ◽

Karl Drlica

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Bovis ◽

Lethal Dose ◽

Clinical Isolates ◽

Methoxyl Group ◽

Human Macrophage ◽

Wild Type ◽

Liquid Media ◽

Human Macrophages ◽

Resistant Strains

ABSTRACT When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents.

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Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates in Lithuania

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.6.2009-2011.2003 ◽

2003 ◽

Vol 47 (6) ◽

pp. 2009-2011 ◽

Cited By ~ 39

Author(s):

Daiva Bakonyte ◽

Aurelija Baranauskaite ◽

Jurate Cicenaite ◽

Anaida Sosnovskaja ◽

Petras Stakenas

Keyword(s):

Mycobacterium Tuberculosis ◽

Molecular Characterization ◽

Clinical Isolates ◽

Wild Type

ABSTRACT Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation −15C→T in the promoter of the mabA-inhA operon.

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Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05894-11 ◽

2011 ◽

Vol 56 (3) ◽

pp. 1253-1257 ◽

Cited By ~ 35

Author(s):

J. Werngren ◽

E. Sturegård ◽

P. Juréen ◽

K. Ängeby ◽

S. Hoffner ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Critical Concentration ◽

Gene Mutations ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Wild Type ◽

First Line ◽

Content Type ◽

Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

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Relationship between Pyrazinamide Resistance, Loss of Pyrazinamidase Activity, and Mutations in the pncA Locus in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.9.2317 ◽

1999 ◽

Vol 43 (9) ◽

pp. 2317-2319 ◽

Cited By ~ 48

Author(s):

M. Mestdagh ◽

P. A. Fonteyne ◽

L. Realini ◽

R. Rossau ◽

G. Jannes ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Radiometric Method ◽

Clinical Isolates ◽

Wild Type ◽

Resistant Strains ◽

Resistance Loss

ABSTRACT Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing ofpncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-typepncA might possess alternative resistance mechanisms.

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