Genes whose expression changed in dying cells but not in three other controls were identified. Expression levels were compared between SD and SS, SD and RD, and SD and RS at each time point. Probes were selected against these three comparisons jointly, with multiple testing adjusted p<0.01. Thirty four genes showed a drug-dependent increase in gene expression (Table S4 for PlasmoDB gene number).

Genes whose expression changed in dying cells but not in three other controls were identified. Expression levels were compared between SD and SS, SD and RD, and SD and RS at each time point. Probes were selected against these three comparisons jointly, with multiple testing adjusted p<0.01. Twenty one genes showed a drug-dependent decrease in gene expression (see Table S5 for PlasmoDB gene number).

Genes in resistant clone B1G9 under drug pressure (RD) acted like sensitive clone Dd2 without drug pressure (SS). Expression levels of RS were compared with SS and RD (RS vs SS and RS vs RD) at corresponding time points and correlation coefficient (r) were computed between SS and RD. Probes that met the criteria of p<0.01 for both comparisons and r>0.85 were selected (Table S6 for PlasmoDB gene number).

Early trophozoites (approximately 20 h post invasion) were treated with antifolates (10 nM WR99210 for Dd2 and 0.5 µM pyrimethamine for TM4/8.2) for varying periods. RNA was isolated and converted into labeled cDNA, and hybridized to a 70-mer microarray as described in the Methods section. The expression ratio of antifolate-treated to untreated control (SD/SS) at each time point was plotted. WR99210 data at 3 h, 6 h, 9 h, and 24 h were compared to pyrimethamine data at 2 h, 4 h, 8 h and 24 h.

DNA microarray data was examined, not just from the coding region of the known antifolate target DHFR-TS (Figure 2), but also from the coding sequences for all the known enzymes of de novo pyrimidine and folate metabolism. Each gene was probed with at least two unique oligonucleotide probes (see Table S3 for oligonucleotide sequences). As in Figure 2C, hybridization of fluorescent cDNA originating from parasites treated with WR99210 for various times (red fluorescence) was compared to non-treated parasites (green fluorescence) and represented as R/G ratios on a log2 scale: A black box signifies no change in expression compared to starting trophozoites, a red box represents some over expression and a green box designates a decrease in DHFR-TS RNA, compared to starting trophozoites (time 0 h). Each bar (SD, SS, RD, and RS) represents different combinations of cell clones (S or R) and treatment (D or S), starting from 3 h to 24 h of treatment.

While 10 nM WR99210 is sufficient to commit 50% of the cells to lethality within 6 h, the parasites continue to obey normal metabolic pattern for hypoxanthine uptake and incorporation into DNA (albeit at a lower amplitude) and continue to develop to schizogony for upto 24 h. (A) Rapid decrease in clonal viability of WR99210 exposed Dd2 cells. Trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Washed cells were diluted and plated in 96-well plates (see methods). Control diluted cells revealed about 20 positive colonies starting at 12–16 days. (B) Continued incorporation of hypoxanthine in WR99210-treated cells. Young trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Cells were pulsed directly with radioactive hypoxanthine for 1 h. Incorporation of radioactive hypoxanthine into DNA was measured by precipitation of nucleic acids on glass fiber filters. Maximum incorporations was seen at 4–8 h into trophozoite development. •; Solvent-treated cells, ▪; WR99210-treated cells. (C) Synchronized Dd2 cells at early trophozoite stage were treated with 10 nM WR99210 or 0.1% DMSO (control) for 48 h. Parasites were visualized by light microscopy of Giemsa-stained blood smears. Images at 6 h, 12 h, 24 h, and 30 h of WR99210 treatment are shown. Based on microscopy, the parasites followed normal development up to about 24 h after WR99210 treatment.

Using multiple probes for DNA microarrays, as well as multiple primer sets to amplify different regions of the DHFR-TS cDNA by qRT-PCR, it is shown that mRNA for DHFR-TS is not overexpressed in protective amounts after antifolate treatment. Note that the microarray data color bar has been set at high sensitivity to pick up any small but significant and reproducible changes in expression. (A) Design of the DNA microarray experiment. This simple scheme was repeated twice for each combination of treatments (WR99210 or DMSO solvent) and varying parasite clones (Dd2 vs B1G9) in Seattle. Young trophozoites were split into treated cells (3 h to 24 h), and untreated cells (0 h). After WR99210 treatment at 10 nM concentrations for various durations, RNA was extracted, converted to cDNA, fluorescently labeled, and hybridized against RNA from pooled reference untreated cells (0 h). One, duplicates from the same biological samples were run with dye inversion. Two, the exact scheme was repeated with independently grown parasites, thus generating 4 hybridizations per time point of WR99210 treatment. This scheme was labeled SD, for Sensitive cells treated with the drug analog WR99210. Finally, the whole scheme of duplicates, was repeated 3 more times (Sensitive clone Dd2 with Solvent (SS), Resistant clone B1G9 with the drug analog WR99210 (RD), and Resistant clone B1G9 with Solvent (RS). Altogether this resulted in microarray data generated from over 123 slides, each carrying about 7,685 oligonucleotide probes. Details of the large experiment are presented in the Methods section. (B) Illustration of regions of DHFR-TS gene queried with oligonucleotide probes (black plus blue boxes) and qRT-PCR primers (black boxes) to measure DHFR-TS mRNA levels in parasites treated with the antifolate WR99210. The DNA sequences corresponding to the 12 unique microarray oligonucleotide probes for DHFR-TS are presented in Table S1. The DNA sequences for the primers used for DHFR-TS qRT-PCR are presented in Table S2. (C) WR99210-treated sensitive cells do not increase DHFR-TS RNA to high levels, as judged by DNA microarrays data that was collected and averaged from 12 different, unique DNA microarray probes representing different parts of the complete DHFR-TS coding region (see Figure 3B). Hybridization of fluorescent cDNA samples originating from parasites treated with WR99210 for various times (red fluorescence) were compared to non-treated parasites (green fluorescence) and represented as R/G ratios on a log2 scale: A black box signifies no change in expression compared to starting trophozoites, a red box represents some over-expression and a green box designates a decrease in DHFR-TS RNA, compared to starting trophozoites (time 0 h). Missing data is shown as gray. Each bar (SD, SS, RD, and RS) represents different combinations of cell clones (S or R) and treatment (D or S), starting from 3 h to 24 h of treatment. (D) Individual DHFR-TS probes revealed no significant differences in RNA levels from within the DHFR-TS coding region. (E) WR99210-treated sensitive cells do not increase DHFR-TS RNA to high levels as judged by qRT-PCR (3–24 h treatment). To independently measure the magnitude of DHFR-TS RNA levels in antifolate treated Dd2 cells, qRT-PCR was applied to 4 different regions of the DHFR-TS coding mRNA sequence. The primer pairs from around nucleotide 13, 135, 1409, and 1542 of the coding region, confirmed that during the 24 h maturation of DHFR-TS, solvent-treated parasites showed a gradual decrease in DHFR-TS RNA. Furthermore, while the qRT-PCR revealed a slightly higher level of DHFR-TS RNA in WR99210-treated Dd2 cells at 24 h, this was not due to an increase over the RNA present before initiation of drug treatment. DHFR-TS mRNA level are represented for solvent-treated cells at 9 h and 15 h (gray bar), for solvent-treated cells at 24 h (light-green bar), for WR99210-treated cells at 9 h and 15 h (black bar), and for WR99210-treated cells at 24 h (dark-green bar). Data are shown as means±SEM. (F) DHFR-TS RNA, probed across the whole coding region, failed to show up-regulation in WR99210-treated Dd2 cells at 24 h. There was a very small but consistent and significant failure to undergo normal repression at 24 h. However, the failure to overexpress protective quantities of DHFR-TS was consistent with the DNA microarray data. DHFR-TS mRNA level in solvent-treated cells (light-green bar) and in WR99210-treated cells (dark-green bar) are shown as means±SEM.