Total GSH levels in blood stages of <i>P</i>. <i>berghei</i> parasites.

<p>GSH levels were determined by HPLC in extracts of purified blood stage <i>P</i>. <i>berghei</i> parasites obtained from asynchronous infections of <b>(A)</b> ANKA wild type, <i>pbggcs-oe1</i> and <i>pbggcs-oe2</i>, <b>(B)</b> N clone (drug sensitive), RC (selected for CLQ resistant) and N/1100 (selected for MFQ resistant) parasites. GSH concentration was significantly increased in the <i>pbggcs-oe</i> parasites as compared to ANKA wild type. Similarly, the drug resistant lines RC and N/1100 displayed significantly higher GSH levels as compared to the sensitive one (N clone). Numbers on boxes represent the mean concentration of GSH. Asterisks denote significant changes in GSH concentration as determined by a One-way ANOVA with Tukey’s Multiple Comparison Test (* = P<0.05, ** = P<0.001).</p

Kaplan-Meir survival curve of mice infected with <i>P</i>. <i>berghei</i> parasites lacking or overexpressing the <i>pbggcs</i> gene.

<p>Mice infected with wild type (green, n = 15), <i>pbggcs-ko</i> (red, n = 10) or <i>pbggcs-oe</i> parasites (blue, n = 10) were treated with intramuscular doses of 20 mg/kg of DHA beginning 1 hr post infection, and daily for four consecutive days. Mice survival was monitored after the 4^th day of DHA treatment (day 0). Mice mortality is defined as mice humanly euthanized due to distress caused by severe malaria. Survival of mice infected with wild type parasites was comparable to the survival of mice infected with <i>pbggcs-oe</i> parasites and rapidly declined after treatment with DHA. In contrast, 90% of the mice infected with <i>pbggcs-ko</i> parasites survived after the DHA treatment. Asterisk denote significant changes in survival as determined by a Log-rank (Mantel-Cox) Test (* = P<0.0001).</p

Drug response curves to CLQ and ART.

<p>Dose response curves show percent growth for each parasite clone relative to the untreated control on day 4 of the assay (5^th day post infection) versus drug concentration. The 4-day suppressive test was carried out for CLQ (A and C) and ART (B and D) on two independent <i>pbggcs-ko</i> (<i>pbggcs-ko1; pbggcs-ko2</i>) and <i>pbggcs</i>-oe (<i>pbggcs-oe1; pbggcs-oe2</i>) clones. To compare the drug responses from the mutant parasites, the parasitemia from each parasite line was normalized to the parasitemia of the untreated control. No significant differences in the EC₅₀ values were observed between parasites with the <i>pbggcs</i> gene silenced or overexpressed as compared to wild type control. Bars represent standard deviation of the mean.</p

Generation of <i>pbggcs</i> overexpression mutants in <i>P</i>. <i>berghei</i>.

<p><b>A.</b> Diagram of the <i>pbggcs</i> overexpression plasmid (Top), the <i>dssurrna</i> genomic locus (Middle) and the predicted integration event of the <i>pbggcs-oe</i> vector at the <i>dssurrna locus (chromosomes 5/6)</i> via single cross-over recombination (Bottom). Open arrows, <i>pbggcs</i> coding region; light grey boxes, <i>P</i>. <i>berghei</i> elongation factor <i>1a promoter;</i> white boxes, 3’UTR <i>pbggcs</i>; dark grey boxes, <i>P</i>. <i>berghei dhfr-ts</i> upstream and downstream regions; black arrows, <i>T</i>. <i>gondii dhfr-ts</i> coding region; hatched boxes, <i>dssurrna</i> DNA sequence<i>;</i> small arrows; positions of primer pairs for PCR analysis; dotted line, predicted size of PCR products; S, SacII; A, ApaI. <b>B)</b> Southern blot analysis of FIGE separated chromosomes of <i>pbggcs-oe</i> parasites. Correct integration of the <i>pbggcs-oe</i> plasmid to the <i>dssurrna</i> integration site was confirmed by incubating the chromosome blots with a <i>P</i>. <i>berghei</i> 3’UTR <i>dhfr-ts</i> specific probe that hybridizes to chromosomes 7 (endogenous <i>dhfr-ts</i>) and chromosome 5/6 (<i>dssurrna</i> integration site). <b>C)</b> PCR analysis confirming integration of the <i>pbggcs</i> over-expression constructs. Primers upstream and downstream from the endogenous <i>dssurrna</i> integration site (primers 211 and 212), to the <i>T</i>. <i>gondii dhfr-ts</i> selection cassette (primers 214) and to the integrated <i>pbggcs</i> sequence (primer 213) were used to verify integration into the <i>dssurrna</i> locus. Products of the predicted size (4.5 kb for primers 211/214 and 2.6kb for primers 212/213) were obtained for both clones. Lanes: m: 100 bp ladder (New England Biolabs), a: primers 211/214, b: primers 212/213.</p