Abstract
The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested early in the ring-to-trophozoite transition in the erythrocytic cycle following gene excision. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway.
Highlights
The repair plasmid used for the manipulation contains a modified selection-linked integration (SLI) region [32] which allows selection for integration events with neomycin
By the end of cycle 1, only arrested pycnotic forms were observed which did not develop further upon prolonged incubation. These results suggested that ablation of PMV expression causes a severe growth defect and developmental arrest between ring and trophozoite stage. Whilst these results showed that truncation of P. falciparum plasmepsin V (PfPMV) affected parasite development in cycle 1, it did not rule out the possibility of defects in egress and invasion of fresh erythrocytes at the end of cycle 0
We have shown for the first time that functional ablation of the pfpmv gene leads to a block at the ring-to-trophozoite transition and failure of the parasite to proliferate
Summary
Diagnostic PCR analysis of DNA extracted from RAP-treated and mock-treated (DMSO-treated control) PMV-C5 parasites confirmed the expected excision event (Fig 2B), a signal indicative of some residual non-excised DNA was detectable, as observed with some previously-described DiCre mutants No schizonts lacking GFP fluorescence could be detected upon exhaustive microscopic examination of the RAP-treated cultures, indicating highly efficient excision of the floxed pfpmv sequence.
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