Figure 4

Fosmidomycin resistance. (a) Fosmidomycin 50% inhibitory concentration (IC₅₀) values (means and standard deviations) were calculated from independent [³H]hypoxanthine assays in duplicate; the number of assays (n) is indicated in parenthesis. Drug susceptibility profiles of Dd2 (wild-type) (n = 7), FOS-R^Dd2-CL1 (n = 5), FOS-R^Dd2-CL2 (n = 3), and control FOS-R^Dd2-CL1-no drug (n = 3). Tests for significant differences between the parental and drug-resistant isolates were performed using analysis of variance with Bonferroni posttests with α = 0.001; significant differences (P value < 0.001) are marked with three asterisks (***). (b) Effect of fosmidomycin on 1-deoxy-D-xylulose 5-phosphate (DOXP) and 2-C-methyl-D-erythritol-4-phosphate (MEP) biosynthesis in intact erythrocyte-free parasites exposed to fosmidoymicin for 24 hours and then labeled with [2-¹⁴C]pyruvic acid as a metabolic precursor. Means and standard deviations were calculated from four independent experiments performed in duplicate. Differences between control (no drug) Dd2 and fosmidomycin-treated Dd2 were significant by Student's t-test for DOXP (1 μmol/l drug: P value 1 × 10^-8; 2 μmol/l drug: P value 1 × 10^-6) and MEP (1 μmol/l drug: P value 4 × 10^-6; 2 μmol/l drug: P value 5 × 10^-8) and are marked with three asterisks (***). (c) To generate the plot, the log₂ ratio of the intensity of each unique probe in FOS-R^Dd2-CL1 was divided by the intensity of each unique probe in Dd2. The probe log ratios were colored by the moving average over a 500 base pair window as indicated in the color bar. The amplification was approximately 100 kb and contained 23 genes including pfdxr (marked with an asterisk). (d) Quantitative RT-PCR analysis of pfdxr transcript level and quantitative PCR analysis gene copy number in FOS-R^Dd2-CL1 compared with Dd2. The means and standard deviations of two independent quantitative RT-PCR and quantitative PCR assays are represented. Significance differences (P value < 0.001) in ΔCt values were determined by Wilcoxon rank sum test and are marked with three asterisks (***).