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.

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

10.1101/2021.05.27.445967 ◽

2021 ◽

Author(s):

Eva Hitz ◽

Natalie Wiedemar ◽

Armin Passecker ◽

Nicolas M. B. Brancucci ◽

Ioannis Vakonakis ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Blood Stage ◽

Targeted Mutagenesis ◽

Missense Mutations ◽

Dependent Protein Kinase ◽

Gene Encoding ◽

Upstream Regulator ◽

Dependent Protein ◽

Kinase A

Cyclic AMP (cAMP) signalling is crucial for the propagation of asexual malaria blood stage parasites. Recent work on Plasmodium falciparum demonstrated that phosphorylation of the invasion ligand AMA1 by the catalytic subunit of cAMP-dependent protein kinase A (PfPKAc) is an essential step during parasite invasion into red blood cells. However, the exact mechanisms regulating PfPKAc activity are only partially understood and PfPKAc function has not been extensively studied in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown mutant, we confirm the essential role for PfPKAc in erythrocyte invasion and demonstrate that PfPKAc is involved in regulating gametocyte deformability. Interestingly, we observed that the conditional overexpression of PfPKAc also caused a profound lethal phenotype by preventing intra-erythrocytic parasite multiplication. Whole genome sequencing of parasites selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the putative parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we show that PfPDK1 is essential for PfPKAc activation, most likely by phosphorylating T189 in the PfPKAc activation loop. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and identify PfPDK1 as a crucial upstream regulator in this pathway and potential new drug target.

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