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

Abstract

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.

Functional inactivation of Plasmodium falciparum glycogen synthase kinase GSK3 modulates erythrocyte invasion and blocks gametocyte maturation

“…This is in line with previous work showing that a hierarchical PfPKA-and PfGSK3βmediated phosphorylation of the cytoplasmic domain of AMA1 is a prerequisite for efficient red blood cell invasion (7)(8)(9)(10)16) and that the use of kinase-specific inhibitors blocks this essential process (8,10,36). In contrast to PfPKA, that was shown to be absolutely essential for red blood cell invasion using conditional knockout systems (9,16,19), PfGSK3β does not appear to be as essential for this process (Figures 2A+3C). This partial redundancy of this kinase might be based on i) that GSK3-specific phosphorylation only enhances the efficiency of red blood cell invasion or ii) that another kinase such as PfCK2 can functionally complement PfGSK3β activity.…”

“…The P. falciparum genome encodes for 65 eukaryotic protein kinases ( 22 ), of which various members have already been described to be involved in essential processes, including egress, host cell invasion, or gametocytogenesis ( 17 , 19 , 38 , 53 ). Nevertheless, to date, only one malaria targeting kinase inhibitor has reached the phase of clinical trials: the PI4K-inhibitor MMV390048 was shown to be safe and well tolerated with high antimalarial activity against all stages of the parasitic life cycle except hypnozoites.…”

“…A substantial body of work highlights the role of this enzyme class in the regulation of host cell invasion ( 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ). For instance, the physiological role of protein kinase A, casein kinase 2 (CK2) as well as glycogen synthase kinase 3 (GSK3) during the host cell invasion has been demonstrated ( 6 , 7 , 8 , 9 , 10 , 16 , 17 , 18 , 19 ). These invasion-related kinases target the cytoplasmic domains of so-called invasins ( 6 , 8 , 9 , 10 , 16 )—proteins that are secreted during the invasion process and mediate binding to surface receptors on the host cell.…”

“…Recent studies provided evidence that protein kinases play an important role in gametocytogenesis and the subsequent formation of gametes ( 17 , 19 , 37 , 38 ). Gametocytes are sexually committed parasite stages that are able to leave the intraerythrocytic proliferation cycle and are transmissible to the mosquito vector ( 39 , 40 ).…”

High-throughput functionalization of theToxoplasmakinome uncovers a novel regulator of invasion and egress

“…The molecular function of all these kinases remains unclear. However, TGGT1_239420 was previously linked to the development of artemisinin resistance in vitro 82 and the Plasmodium falciparum ortholog of TGGT1_268210 was recently linked to regulation of protein kinase A (PKA) 83 .…”