Invasion of hepatocytes by Plasmodium sporozoites requires cGMP‐dependent protein kinase and calcium dependent protein kinase 4

Abstract

Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second-messengers - cGMP mediated by the parasite's cGMP-dependent protein kinase (PKG), and Ca , mediated by the parasite's calcium-dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.

Multiple short windows of calcium-dependent protein kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis

“…Bumped kinase inhibitors such as 1294 appear to have a broad-spectrum anti-protozoal activity by targeting functional orthologues of CDPK4 (Van Voorhis et al, 2017) and this study provides a first insight into the molecular functions of this atypical Ca 2+ -dependent protein kinase. Remarkably, CDPK4 has been previously reported to be important for sporozoite motility (Govindasamy et al, 2016) and oocyst formation (Billker et al, 2004), two processes unrelated to male gametogenesis. Similarly, in the related apicomplexan parasite Toxoplasma gondii , the functional orthologue of CDPK4 (TgCDPK1) is essential for host cell egress and invasion (Lourido et al, 2010).…”

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase

“…Specifically, the P. falciparum cGMPdependent kinase (PfPKG) plays a major role in both replication and transmission of the Plasmodium parasite, including exflagellation and gametogenesis in the asexual blood stage, invasion of hepatocytes by sporozoites, and gliding motility of ookinetes (5)(6)(7)(8)(9). Hence, PfPKG has emerged as a promising target for development of antimalaria therapies complementary to current malaria drugs, whose effectiveness is limited by increasing resistance (2,10,11).…”

Gliding motility powers invasion and egress in Apicomplexa

“…Several other proteins and molecules have been linked to signalling pathways that regulate the glideosome. cGMP-dependent protein kinase (PKG) triggers microneme secretion and parasite egress in a calcium-independent manner 153,[157][158][159] . Recently, phosphatidic acid has been identified as an important lipid mediator of microneme secretion that acts in parallel to calcium 160 .…”