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.

Fig. 1. Schematic maps of pUF-1 and its parental plasmid pCC4

In pUF-1 a modified yDHODH gene replaces the bsd gene present in the parental plasmid pCC4 (see Materials and Methods). Hsp86 5′ = 5′ UTR of PfHSP86 gene; yFCU = yeast cytosine deaminase-uracil phosphoribosyl transferase fusion gene; Cam 5′ = 5′ UTR of P. falciparum calmodulin gene; PbDT3′ = 3′UTR of P. berghei dihydrofolate reductase-thymidylate synthase gene; Hrp2 3′ = P. falciparum histidine rich protein 2 3′ UTR; bsd = blasticidin S deaminase; yDHODH = yeast dihydroorotate dehydrogenase; pGEM3z = pGEM3z plasmid (Genbank accession X65306) backbone containing an ampicillin resistance gene (Amp^R)

Fig. 2. Schematic diagram of the knockout strategy for the VP2 gene

The VP2 locus on P. falciparum chromosome 12, the pUF-1 derived knock out plasmid (pUF1ΔVP2) used to disrupt the endogenous VP2 gene and the predicted VP2 locus after double cross over integration are shown. The binding sites of primers used in PCR reactions to confirm the knock out are indicated by small arrows labeled a, b. The location of the probe and the expected sizes of the fragments detected by Southern blot analysis of genomic DNA digested with EcoRI (E) and HindIII (H) are shown.

Fig. 3. Confirmation of VP2 gene knockout

A) Diagnostic PCR. Genomic DNA isolated from wild type and transgenic parasites served as template for PCR reactions using primers VP2 Sense5′ and VP2 Antisense3′ (Table 2; positions indicated in Fig 2). The PCR produced 4.6 kb and 2.5 kb products with knock out and wild type genomic DNA, respectively. M- Marker DNA fragments (1kb, Promega), WT- Wild type, KO- VP2 knock out. B) Southern blot analysis of genomic DNA from D10 wild type and D10 VP2 knock out parasites digested with HindIII or EcoRI. The probe, generated from a 3′ segment of the VP2 gene (position shown in Fig. 2), hybridized to 5.8 kb (HindIII) and 10 kb (EcoRI) fragments of wild type DNA, and to 7.3 kb (HindIII) and 7.6 kb (EcoRI) fragments of transgenic DNA.

Fig. 4. yDHODH transgenic PfVP2 knockout parasites are susceptible to the compounds used for other selectable markers

Growth inhibition of wild type D10 and ΔVP2::yDHODH transgenic parasites was assessed by standard ³H-hypoxanthine incorporation assay for atovaquone (A), DSM1 (B), WR99210 (C), and blasticidin (D). The calculated IC₅₀ values for these compounds are listed in the Results section. Solid lines, wild type D10 parasites; dashed lines, ΔVP2::yDHODH parasites.