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

Abstract

The protozoon Toxoplasma gondii, an opportunistic human pathogen, secretes organelles called micronemes, which play a key role in parasite motility, host-cell invasion and egress. This process is now shown to involve the T. gondii calcium dependent protein kinase 1 (TgCDPK1). The lack of this kinase family in mammalian hosts makes this protein an attractive target for drug design. Toxoplasma gondii is an opportunistic human pathogen that secretes organelles called micronemes during infection. This is important for parasite motility, host-cell invasion and egress. It is now shown that the secretion of micronemes is dependent on the T. gondii calcium-dependent protein kinase 1. This kinase is not found in the parasite's mammalian hosts, and might represent a valid drug target. 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 various cellular functions such as plasma membrane repair in plants and animals2,3, the discharge of defensive spikes in Paramecium4, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons5. In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion1,6,7. Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show 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. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite’s life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase 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 have a function in calcium-regulated secretion in other organisms. Because 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.