High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac

Abstract

Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran transposon piggyBac, transformed parasites by integration into the P. falciparum genome in the presence of a transposase-expressing helper plasmid. Multiple integrations occurred at the expected TTAA target sites throughout the genome of the parasite. We were able to transform P. falciparum with this piggyBac element at high frequencies, in the range of 10(-3), and obtain stable clones of insertional mutants in a few weeks instead of 6-12 months. Our results show that the piggyBac transposition system can be used as an efficient, random integration tool needed for large-scale, whole-genome mutagenesis of malaria parasites. The availability of such an adaptable genetic tool opens the way for much needed forward genetic approaches to study this lethal human parasite.

Fig. 1.

Plasmid design and procedure for piggyBac transformation of P. falciparum. (A) pXL-BACII-DHFR, a piggyBac transposon vector for transformation of P. falciparum, was created by cloning the hdhfr coding sequence under the control of 5′ calmodulin and 3′ histidine-rich protein-2 into the minimal piggyBac vector pXL-BACII. The hdhfr cassette was excised from the vector pHH1 as a 2.2-kb EcoRI/BglII fragment and cloned into pXL-BacII, such that it is flanked by the piggyBac ITR 1 and ITR 2. (B) The pHTH helper plasmid was constructed by cloning the piggyBac transposase coding sequence under the control of P. falciparum 5′ and 3′ hsp 86 sequences. piggyBac transposase expressed in the blood stages of P. falciparum will catalyze the transposition of the piggyBac element from the vector pXL-BACII-DHFR into the P. falciparum genome. (C) Schematic representation of the procedure used to transform P. falciparum. Mature blood-stage parasites were purified by passage through a magnetic column (Miltenyi Biotec) and reintroduced into culture with erythrocytes preloaded by electroporation with plasmids pXL-BacII-DHFR and pHTH. After one to four generations of growth in the preloaded erythrocytes, parasites were selected with 2.5 nM WR99210 until drug-resistant parasites emerged in culture.

Fig. 2.

Confirmation of piggyBac integration in the P. falciparum genome. (A) Parasite genomic DNA was digested with EcoRI, and Southern blot hybridization using a hdhfr probe identified unique piggyBac hybridization bands from parasites cotransfected with pXL-BACII-DHFR and pHTH. In experiments 1 and 2, parasites were transfected with plasmids pXL-BACII-DHFR and pHTH, in 1:1 and 2:1 ratios, respectively, and the parasites were maintained for one generation in plasmid-loaded erythrocytes before selection with WR99210. In the other experiments (–8), parasites were transfected with the plasmids in a 2:1 ratio and maintained in culture for four generations before selection with WR99210. Transfections for experiments 1, 2, 7, and 8 were initiated in a 5-ml culture volume, and, in experiments 3–6, transfections were performed in 200-μl cultures in a 96-well plate. After Southern blot hybridization, the episomal band was seen as a 6.2-kb fragment. The identified piggyBac integrations are indicated by letters. (B) Southern blot hybridization analysis of individual clones obtained from populations 1 and 2 identified clones with different sites of integrations. Clones A1, B8, B12, C8, and F4 appear to have the common insertion “a” and are likely to be of the same origin. Clone B4 and G5 have dissimilar sites of integration, “b” and “c.”

Fig. 3.

Identification of piggyBac integration sites in the P. falciparum genome. Inverse PCR analysis was used to identify the piggyBac 5′ TR insertion sites. Briefly, genomic DNA from drug-resistant populations were digested with either Sau3AI or RsaI and self-ligated in a dilute reaction. Sau3AI self-ligated fragments were digested with TseI to remove the episomal fragment. The remaining self-ligated fragments were used as templates in an inverse PCR to identify sites of integration into the genome. Sequence analysis identified nine different sites of integration in eight different chromosomes, suggesting a genome-wide insertion of piggyBac. As expected, the piggyBac element had inserted in a TTAA target sequence in all of the analyzed clones. PCR analysis was then performed by using a genomic primer at each insertion site and a primer in ITR1 to confirm that the insertion of the piggyBac element was complete. Further sequence analysis confirmed the insertion of piggyBac ITRs into a TTAA target sequence that resulted in the duplication of the target site in the genome. The italicized sequences in insertions b, g, h, and i were confirmed by Southern blot hybridization analyses (data not shown).