EtaA and EtaR control ETA susceptibility. MSm pMH29 clones expressing EtaA (lanes a), vector control (lanes b), and EtaR (lanes c) are shown spotted in 10-fold decreasing dilutions (from top to bottom) onto 7H11 plates containing the indicated concentration of drugs (in μg/ml). In a microbroth dilution assay the MICs (in μg/ml) for ETA were: lane a, 0.78–1.56; lane b, 50; and lane c, 200 +; for ETA S-oxide: lane a, 0.10–0.20; lane b, 0.78–1.56; and lane c, 6.25–12.5; and for INH: lane a, 1.56–3.13; lane b, 1.56–3.13/6.25, and lane c, 1.56–3.13.

EtaA-associated mutations and cross-resistance in patient isolates from Cape Town, South Africa. (A) Thiacetazone (11) and thiocarlide (12). (B) Map of mutations in EtaA found in patient isolates resistant to ETA and thiacetazone. Chromosome coordinates and gene designations are in reference to the sequenced genome of MTb strain H37Rv. (C) Cross-resistance determination of patient isolates and the associated nucleotide and amino acid alterations observed. Drug susceptibility for ETA is reported as: susceptible (S) if the culture failed to grow at 2.5 μg/ml, low-level resistant (L) if weak growth was observed at 2.5 μg/ml, moderately resistant (M) if strong growth was observed 2.5 μg/ml, and high-level resistant (H) if growth was observed at 10 μg/ml. Drug susceptibility for thiacetazone (TA)/thiocaslide (TC)/INH is reported as susceptible (S) if the culture failed to grow at 0.5 μg/ml, low-level resistant (L) if weak growth was observed at 0.5 μg/ml, moderately resistant (M) if weak growth was observed at 2.0 μg/ml, and high-level resistant (H) if strong growth was observed at 2.0 μg/ml.

In vivo production of (2-ethyl-pyridin-4-yl)methanol (5) from ETA by whole cells of MTb. A culture of MTb strain H37Rv at OD₆₅₀ of 1.0 was concentrated 10-fold in 7H9 media, and [1-¹⁴C]ETA at 0.01 μg/ml was added. (A) After incubation at 37°C metabolism of [1-¹⁴C]ETA was visualized by TLC and autoradiography. Lanes a–h correspond to sequential filtered culture samples taken at 0.2, 0.25, 0.75, 1.5, 2.5, 5.0, 8.5, and 25 h, respectively. Lane i represents media autooxidation after 25 h of incubation without bacterial cells. The metabolites observed cochromatographed with commercial and characterized synthetic samples of ETA (1), ETA S-oxide (2), ETA nitrile (3), and ETA amide (4). (B) Mycobacteria from the same sequential culture aliquots (500 μl) were collected by filtration onto 0.22-μm filter disks under vacuum, they were washed twice with PBS (500 μl), and the cell-associated radioactivity was measured. (C) The reversed-phase HPLC retention time of the unknown major metabolite (5) was used to guide cold large-scale ETA feeding experiments where we isolated unlabeled metabolite that gave a mass of 137 (137.9 MH⁺). We assigned this as (2-ethyl-pyridin-4-yl)methanol and confirmed the identity of (5) by cochromatography with a synthetic characterized alcohol standard. The upper HPLC continuous radiodetector spectrum corresponds to A lane i, media control and the lower spectrum; lane d, time point 1.5 h, where the UV₂₅₄ trace of (2-ethyl-pyridin-4-yl)methanol is superimposed in gray.

EtaA and EtaR control ETA metabolism. (A) The MSm clones used in Fig. 2 were analyzed for their ability to metabolize [1-¹⁴C]ETA. Lanes a–f correspond to sequential filtered culture samples taken at 0, 30, 90, 180, 330, and 900 min, respectively. Metabolites were identified as in Fig. 1 and labeled accordingly. (B) The cell-associated radioactivity for each sequential clone aliquot (1,000 μl) was determined as in Fig. 1B. ■, MSm overexpressing EtaA; ●, wild-type MSm; ▴, MSm overexpressing EtaR. (C) Macromolecule-associated radioactivity was calculated by resuspending microcentrifuged 900-min aliquots (400 μl) in PBS, bead beating (3 × 45 sec), and extensively dialyzing the total lysates to remove molecules smaller than 10 kDa before scintillation analysis. Column 1, MSm overexpressing EtaA; column 2, wild-type MSm; column 3, MSm overexpressing EtaR.

The proposed activation process for thioamide oxidation of ETA.