Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma

Abstract

Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge1. This process regulates diverse cellular functions like plasma membrane repair in plants and animals2,3, discharge of defensive spikes in Paramecium4, and secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons5. In animal cells, serine/threonine kinases including PKA, PKC and CaM-kinases have been implicated in calcium-signal transduction leading to regulated secretion1,6,7. Although plants and protozoa also regulate secretion via intracellular calcium, the means by which these signals are relayed have not been elucidated. Here we demonstrate that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. This phenotype was recapitulated using a chemical biology approach, wherein pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, since expression of a resistant kinase mutant reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates8, suggesting that related CDPKs may play a role in calcium-regulated secretion in other organisms. Since this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.