Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection

Abstract

Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.

Inhibition of Cytochrome bc1 as a Strategy for Single-Dose, Multi-Stage Antimalarial Therapy

“…Like atovaquone and the parent ELQs, ELQ-400 was inactive against transgenic parasites expressing yeast dihydroorotate dehydrogenase (DHODH), showing that its predominant mechanism of action involved pyrimidine starvation secondary to cyt bc 1 inhibition. 19 However, in contrast to ELQ-121, which showed a nearly 1,000-fold loss of potency against atovaquone-resistant Tm90-C2B parasites (which contain a Y268S point mutations in cyt b) ELQ-400 retained substantial activity against this strain with an IC 50 of 35nM.…”

“…One such target is Complex III of the mitochondrial electron transport chain, which is also known as the cytochrome bc 1 complex (cyt bc 1 ) and is essential for pyrimidine biosynthesis in Plasmodium. 19,20 Several cyt bc 1 inhibitors, including the naphthoquinone atovaquone 17 and the 4(1H)-quinolone ELQ-300, 18 are potent single-dose prophylactics, with remarkable multi-dose efficacy against blood-stage malaria parasites. 18,21 Although it has not yet been possible to formulate these compounds as singledose, blood-stage therapies, it is likely that this limitation is related to poor solubility, which restricts the plasma concentrations that can be achieved after a single oral dose.…”

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

“…Although the number of selectable markers increased (143)(144)(145) and alternate transfection methods were developed (146), genetic manipulation techniques and efficiency did not change drastically for P. falciparum, aside from the introduction of the "double crossover" technique (Fig. 6B).…”

ATP Synthase Complex of Plasmodium falciparum

“…We used the classical gene disruption approach, which involves the attempted replacement of all or part of the gene by double crossover recombination. To decrease the possibility that our experimental results could be due to an unknown interaction of the target gene product with the pathway blocked by the selective agent or other artifact, we transfected parasites with two different disruption plasmid constructs for each gene, one selected using the antifolate WR99210 and one selected by DSM1 (26,28), which blocks the pyrimidine biosynthesis pathway (Fig. 3A).…”