The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.Cyclic GMP (cGMP)-dependent protein kinases (PKGs) are the major intracellular mediators of cGMP signal transduction in eukaryotic cells. Mammalian PKGs regulate a number of physiological processes including smooth muscle relaxation, platelet aggregation, intestinal secretion and hippocampal and cerebellar learning (reviewed in reference 27). Unlike cAMP-dependent protein kinase (PKA), PKG comprises both a catalytic domain and a regulatory domain in a single polypeptide. In the inactivated state, part of the regulatory domain (a substrate-like sequence known as the autoinhibitory domain) is thought to be bound to the catalytic domain. This binding is released and the enzyme activated upon binding of cGMP to the regulatory domain and opening of the substrate-binding region within the catalytic domain (1, 14). The PKG of the malaria parasite Plasmodium falciparum (PfPKG) and orthologues from the related coccidian parasites Toxoplasma and Eimeria are encoded by a single-copy gene and have been shown to differ from the mammalian enzymes in several respects. These include a larger number of cGMP binding sites (three functional plus one degenerate) in the regulatory domain of the apicomplexan enzymes (6, 7, 17), highly cooperative stimulation by cGMP (8, 17), and relative insensitivity to 8-substituted cGMP analogues (6, 7, 26). The parasite isoforms also lack an N-terminal leucine zipper motif that mediates homodimerization in mammalian isoforms, and evidence suggests that they are monomeric (17). Interestingly, it has been shown that the coccidian PKGs exist as both cytosolic and membrane-associated (mediated by N-terminal myristoylation and palmitoylation) isoforms (12), but the amino acid motifs required for these modifications are absent in the PfPKG sequence, and it seems to lack acylated forms (8).The coccidian PKGs have been shown to be the target of a potent anticoccidial agent, 4-[2-(4-fluorphenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl]pyridine (compound 1), which is a competitive inhibitor of ATP binding (17). The selectivity of the compound for apicomplexan enzymes is attributed to the relative accessibility of a hydrophobic pocket that overlaps with the ATP binding site conferred by a critical threonine residue in the key “gatekeeper” position (T761 in Toxoplasma gondii; T770 in Eimeria tenella) (11). Access is thought to be prevented by the relatively bulky side chain of the residue that occupies the corresponding position in mammalian PKGs. Expression of compound 1-insensitive coccidian PKGs (harboring T/Q or T/M gatekeeper residue substitutions) in Toxoplasma abolished the effects of the inhibitor on the parasite, thus establishing that PKG is the primary target of compound 1 in T. gondii. These compound 1-insensitive mutants were used to determine a role for coccidian PKG in secretion of micronemal adhesive proteins, attachment to and invasion of host cells and gliding motility of E. tenella sporozoites and T. gondii tachyzoites. Compound 1 has also been shown to be a potent inhibitor of the native P. falciparum PKG enzyme (50% inhibitory concentration [IC₅₀] of 8.53 nM on cGMP-dependent kinase activity in purified fractions using a fixed ATP concentration of 5 μM in the assay) but has limited in vivo activity in the rodent malaria model P. berghei (8).P. falciparum causes the most serious form of malaria in humans and is responsible for the deaths of approximately one million people each year. The parasite life cycle is complex consisting of distinct phases in the mosquito vector and the human host. Pathogenesis is caused by asexual parasites which proliferate within red blood cells. The invasive form (merozoite) enters a red blood cell and forms a ring-stage parasite. This develops into a trophozoite which feeds on hemoglobin, and the resulting schizont releases up to 32 daughter merozoites in a 48-h cycle. A small proportion of the schizonts release merozoites that differentiate into male or female gamete precursors (gametocytes). These sexual cells are essential for malaria transmission and must be taken up by an Anopheles mosquito to continue the life cycle. Upon entering the insect midgut, environmental cues trigger gametogenesis. Recently, we reported that PfPKG plays an essential role in initiating this process. The initial morphological change after activation of P. falciparum gametocytes, from crescent-shaped to spherical (known as rounding up), is inhibited by compound 1. Genetically manipulated parasites expressing a mutant (T618Q gatekeeper substitution) compound 1-insensitive but fully active PKG, introduced by allelic replacement, were resistant to the effects of compound 1 on gametogenesis, demonstrating a direct role for the enzyme in the initiation of this process (23).There is also evidence suggesting a role for PKG in P. falciparum erythrocytic stages. It has been reported that PfPKG is expressed at both the mRNA and the protein level in asexual blood stages, as well as the sexual phase of the life cycle (6, 8). Although the PfPKG gene could be targeted for allelic replacement, we were unable to disrupt it, suggesting an essential role for PKG in P. falciparum asexual blood stage development since it is this life cycle stage on which transfection and drug selection of genetically modified parasites are performed. Furthermore, we and others (8, 23) have observed that compound 1 inhibits in vitro-cultured P. falciparum (IC₅₀s in the sub-low-μM range depending on the isolate). Although the role of PKG in the initiation of gametogenesis has been elucidated, its role in erythrocytic stages was unknown prior to the present study.We demonstrate here that PfPKG has a central role in the late stages of erythrocytic schizogony. Compound 1-treated P. falciparum parasites develop normally through the trophozoite stage but arrest late in schizont development, forming large dysmorphic schizonts. Genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG are resistant to the effects of both compound 1 and (a second imidazopyridine PKG inhibitor) compound 2 on schizogony but not to the broad-specificity serine/threonine kinase inhibitor staurosporine. This demonstrates that PfPKG is the primary target of the inhibitors and provides direct evidence that this enzyme is essential for progression of the asexual erythrocytic stage of the malaria parasite life cycle.