Article Figures & Data
Figures
- Fig 1
Dose-response effects of ISO on mycolic acid biosynthesis in wild-type and HadABC-overexpressing M. tuberculosis strains. The inhibitory effect on the incorporation of [1,2-14C]acetate was assayed by labeling in the presence of increasing drug concentrations. The corresponding fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were extracted. Equal counts (20,000 cpm) were loaded onto a TLC plate, and lipids were developed twice in hexane-ethyl acetate (19:1, vol/vol) and exposed overnight to a film.
- Fig 2
Mutations within HadA or HadC confer growth resistance during ISO treatment. Mid-log-phase cultures of wild-type (WT) M. tuberculosis and strains carrying either the C61G mutation in HadA (MTTR3) or the V85F mutation in HadC (MTTR18) were streaked onto Middlebrook 7H10 OADC plates supplemented with increasing concentrations of ISO (1, 2.5, and 5 μg/ml). Plates were incubated for 2 to 3 weeks at 37°C, after which growth was visualized.
- Fig 3
Mechanisms of action and/or markers of resistance to the major antitubercular thiocarbamide-containing prodrugs. ETH, TAC, and ISO are all prodrugs that get into tubercle bacilli by passive diffusion, where they are activated by the monooxygenase EthA. Once activated, ETH inhibits mycolic acid biosynthesis by targeting the FAS-II enoyl ACP reductase InhA. Activated TAC and ISO affect mycolic acid composition through InhA-independent mechanisms involving other important mycolic acid biosynthetic enzymes. TAC has been shown to inhibit mycolic acid cyclopropanation (CMAS), and high resistance levels were found to be associated with mutations within the methyltransferase MmaA4 and within the HadA or HadC component of the FAS-II dehydratase. This study provides evidence that the same mutations in HadA or HadC correlate with increased levels of resistance to ISO, suggesting that TAC and ISO can have mechanisms of activation and action in common. In addition, oleic acid synthesis appears to be specifically inhibited through inhibition of the stearoyl-CoA desaturase DesA3 by activated ISO.
Tables
- Table 1
Antimycobacterial activities of INH, ETH, and ISO against M. bovis BCG strains overexpressing various FAS-II components
Strain MIC99 (μg/ml)a INH ETH ISO pMV261 0.1 2.5–5 0.5–1 pMV261::kasA 0.1 5 0.5–1 pMV261::inhA 2 50 1 pMV261::mabA 0.1 5 0.5–1 pMV261::hadABC 0.1 2.5–5 5 ↵a MIC99s were determined by dilution on 7H11 solid agar medium supplemented with OADC.
- Table 2
Antimycobacterial activities of TAC and ISO against M. tuberculosis strains overexpressing various Had complexes
Strain MIC99 (μg/ml)a TAC ISO pMK1 0.25 0.5 pMK1::hadAB 2.5 2.5 pVV16 0.25 0.5 pVV16::hadBC >25 1 pMV261::hadABC >50 10 ↵a MIC99s were determined by dilution on 7H10 solid agar medium supplemented with OADC.
- Table 3
Antimycobacterial activity of ISO against M. tuberculosis strains harboring mutations within hadA or hadC
Strain ISO MIC99 (μg/ml)a Wild type 0.5–1 MTTR3 (HadA_C61G) >10 MTTR6 (HadA_C61S) >10 MTTR2 (HadC_T123A) 10 MTTR18 (HadC_V85F) >10 ↵a MIC99s were determined by dilution on 7H10 solid agar medium supplemented with OADC.
