Abstract
Malaria remains a heavy global burden on human health, and it is important to understand the molecular and cellular biology of the parasite to find targets for drug and vaccine development. The mouse malaria model is an essential tool to characterize the function of identified molecules; however, robust technologies for targeted gene deletions are still poorly developed for the widely used rodent malaria parasite, Plasmodium yoelii. To overcome this problem, we established a DiCre-loxP inducible knockout (iKO) system in P. yoelii, which showed more than 80% excision efficacy of the target locus and more than 90% reduction of locus transcripts 24h (one cell cycle) after RAP administration. Using this developed system, cAMP-dependent protein kinase (PKAc) was inducibly disrupted and the phenotypes of the resulting PKAc-iKO parasites were analyzed. We found that PKAc-iKO parasites showed severe growth and erythrocyte invasion defects. We also found that disruption of PKAc impaired the secretion of AMA1 in P. yoelii, in contrast to a report showing no role of PKAc in AMA1 secretion in P. falciparum. This discrepancy may be related to the difference in the timing of AMA1 distribution to the merozoite surface, which occurs just after egress for P. falciparum, but after several minutes for P. yoelii. Secretions of PyEBL, Py235, and RON2 were not affected by the disruption of PKAc in P. yoelii. PyRON2 was already secreted to the merozoite surface immediately after merozoite egress, which is inconsistent with the current model that RON2 is injected into the erythrocyte cytosol. Further investigations are required to understand the role of RON2 exposed on the merozoite surface.
Highlights
Malaria is still a major public health concern in the world [1]
To generate dimerisable Cre-recombinase (DiCre)-expressing P. yoelii, a DNA fragment containing a DiCre expression cassette and a hDHFR-yFCU expression cassette was integrated into the Pyp230p gene locus, which is dispensable in all development stages [27] (Fig. 1A)
immunofluorescent assay (IFA) confirmed expres sion of FRB-Cre60 and FKBP-Cre59 at both the ring and schizont stages of DiCre-expressing P. yoelii clones, which largely colocalized with the nucleus signal (Fig. 1E). quantitative reversetranscription polymerase chain reaction (qRT-PCR) revealed that the amount of cre60 transcripts normalized by those of met-trna synthetase was not reduced for 29 days (9 passages in mice), indicating stable cre60 transcription in the generated clone for at least one month (Fig. S1)
Summary
Malaria is still a major public health concern in the world [1]. The causative pathogens of malaria are obligate intracellular protozoan parasites which belong to the genus Plasmodium and are transmitted by Anopheles mosquitoes. Invasion is mediated by the sequential secretion of molecules from parasite organelles, including micronemes and rhoptries, which have unique roles such as interaction with erythrocyte receptors [2,3,4] Secretion of these mole cules is regulated by intracellular calcium mobilization and phosphor ylation [5,6]. Once the cellular cAMP concentration reaches a threshold level, PKA is activated and phosphorylates Ser610 of P. falciparum AMA1, which is essential for erythrocyte invasion [7,9,10,11] This model was developed by analyzing P. falciparum, which can invade erythrocytes immediately after egress; other malaria parasite species such as P. yoelii and P. knowlesi require several minutes after egress for merozoites to achieve compe tency to invade erythrocytes, indicating that these parasites need to be activated after egress [12]. Understanding this activation process would provide insights to understand the erythrocyte invasion mechanisms of malaria parasites
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