The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

Transcriptional profiling defines dynamics of parasite tissue sequestration during malaria infection.

BACKGROUND: During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes - sexual stage precursor cells - likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the bloodstream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. RESULTS: We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post-invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and approximately 30 hours post-invasion and mature gametocytes after around 7 days post-invasion. CONCLUSIONS: This study provides a bioinformatics resource for the functional elucidation of parasite life cycle dynamics and specifically demonstrates the presence of the gametocyte ring stages in circulation, adding significantly to our understanding of the dynamics of gametocyte sequestration in vivo.

Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen.

An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rγ(null) mice engrafted with human erythrocytes. In contrast to other P. falciparum mouse models, this system generated very consistent results and evinced a dose-response relationship and therefore represents an unprecedented in vivo model for quantitative comparison of the functional potencies of malaria-specific Abs. Our data suggest a role for CyRPA in erythrocyte invasion by the merozoite. Inhibition of merozoite invasion by CyRPA-specific mAbs in vitro and in vivo renders this protein a promising malaria asexual blood-stage vaccine candidate Ag.