Fig 1.
Effect of the addition of the two naphthoquinones, plumbagin and lawsone (a and b) on the dUMP dependent NADPH oxidase activity of MtbThyX (c-f) [25]. The NADPH oxidase activity was assayed by measuring the time-dependent decrease in OD340. The assays were carried out either in the absence or presence of different concentration (μM) of either plumbagin (c,e) or lawsone (d,f). The initial velocities derived from (e and f) obtained were plotted against inhibitor concentrations (g,h).
Fig 2.
a) Determination of Km, Vmax, and IC50. Substrate (dUMP) saturation experiments, were performed, using the 3H release method, either in the absence of plumbagin (0 μM PG) or in its presence (10 μM PG) using Mtb ThyX. The concentrations of the co-substrates, NADPH and methylenetetrahydrofolate were 200 μM in each case. Considering the Km values of ThyX (S5 Fig) for NADPH and methylene tetrahydrofolate, 35.22, and 6 μM respectively, the extent of saturation achieved was 86% for the former and 97%, latter. Curve fitting and derivation of Km and Vmax were done using the Michelis-Menten equation with the help of GraphPad Prism software. b) For the determination of IC50, the activity of Mtb ThyX was assayed in the presence of increasing concentrations of plumbagin (inhibitor) using a saturating concentration of dUMP (40 μM). Curve fitting was done using the formula, Y = Bottom +(Top-Bottom)/ (1+X/IC50), in which Y is the activity of the enzyme at any given inihibitor concentration, X. Top and bottom are plateaus in the units of the Y axis. Each data point represents the mean of three technical replicate experiments ±standard deviation. In some cases the error bars are not visible as they are too small compared to the size of the symbols.
Table 1.
Enzymological attributes of Mtb ThyX.
Fig 3.
Comparison of mycobacterial growth inhibitory effects of plumbagin, lawsone, and rifampicin.
Growth of mycobacteria either Msm (a and b) or Mtb (d) was monitored by measuring either visible growth (OD600) (a and d) or in the case of Msm (b) additionally by colony counting (CFU/ml). The MICs of plumbagin and rifampicin were also determined and compared by performing agar diffusion assay using Msm (c). Error bars in (c) represent the standard deviation from the mean of three replicate assays. The color codes are green, red, and blue for lawsone, plumbagin, and rifampicin, respectively.
Table 2.
MIC/ MBC values (μg/ml) of inhibitors reported in this study.
Fig 4.
Effect of plumbagin (a-c) and rifampicin (d-f) on cell viability and [dTTP]/[dATP] ratio. Survival after plumbagin and rifampicin treatment at the doses mentioned was measured by either OD600 (a and d) or CFU/ml (b and e). The corresponding [dTTP]/[dATP] ratios derived by performing mass spectrometric analysis are shown in (c) and (f). The experiments were performed five times. The complete dNTP profiles from which these ratios were derived are presented in S2 Fig. The results are presented as mean ± SD (standard deviation). Significant differences in the [dTTP]/[dATP] ratios of the treated samples relative to the untreated ones are marked by asterisks, * for p = 0.0104 and ** for p = 0.0078. Significance levels (p values) were determined by performing paired t-tests using Graph Pad Prism software.
Fig 5.
Effect of overexpression of the gene for Mtb ThyX from the inducible vector pLAM12 on the survivability of Msm cells treated with different doses of plumbagin as indicated.
Survivability was measured by counting the CFUs present per ml in the cultures in which ThyX gene expression was either induced or uninduced after incubation for 24 hrs at 37 ±0.5°C. An empty vector control was also included. The actual CFU values are presented in the S1 Table.