Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle

Abstract

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes.

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

“…An alternative experimental design to test this hypothesis would be to employ a promoter swap strategy, such that the FAS-II gene is expressed in sporozoites but not in late liver stages, for example, using the CSP or TRAP promoter that is upregulated during mosquito stages (1). A variation of this strategy would be to express rapamycin-inducible Di-Cre recipient clones of P. falciparum under early-liver-stage-specific promoters to excise FAS-II genes only during liver-stage development (57). An alternative strategy could utilize regulatable tags, which have successfully been used to demonstrate the essentiality of bloodstage expressed proteins (58,59) but, to date, not for pre-erythrocytic stages.…”

Novel Thioredoxin-Like Proteins Are Components of a Protein Complex Coating the Cortical Microtubules of Toxoplasma gondii

“…If the target gene is essential and the knockout mutant fails to grow as a clone, one can still use the mixed population of the Cre-transfected LoxP knock-in line to identify the mutants under the microscope based on fluorescence and analyze the knockout phenotype in live cells. This strategy can be used in any Cre-LoxP-based technique regardless of how Cre expression is controlled (i.e., by transient transfection as described previously [29,30] and in this work or by conditional expression, as in the case of the DiCre system first developed for mouse genetics [46] and recently implemented in T. gondii and P. falciparum [47,48]). …”

Conditional U1 Gene Silencing in Toxoplasma gondii

“…A sequence comprising an array of 10 repeated U1 recognition sequences was placed immediately downstream of the floxed PbDT3' UTR ( Fig 7A). The construct was transfected into the previously-described P. falciparum 1G5DiCre recipient clone [6], which constitutively expresses DiCre from a genomic locus. Rapamycin induced DiCre mediated recombination was predicted to excise the PbDT 3' UTR, concomitantly translocating the 10 U1 recognition sequences to a position directly adjacent to the pfsub1 STOP codon.…”

“…Immunofluorescence assay (IFA) and Western blot IFA was performed on Percoll purified schizonts as described [6] using the anti PfSUB1 mAb NIMP.M7 and the anti-HA mAb 3F10 (Roche). For Western blot analysis, purified schizonts were solubilised in SDS sample buffer and fractionated on a 4-15% Biorad Mini PROTEAN Stain-free gradient SDS polyacrylamide gel.…”