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 surge. This process regulates various cellular functions such as plasma membrane repair in plants and animals, the discharge of defensive spikes in Paramecium, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons. 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 secretion. 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 ciliates, 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.

Figure 1. TgCDPK1 is essential for the lytic cycle

a, Regulatable, HA9-tagged TgCDPK1 was added to wild-type (WT) to create a merodiploid (mDip). Endogenous TgCDPK1 was replaced with phleomycin resistance (ble^R) to generate the cKO. Complementation with c-Myc tagged mutant alleles (denoted by cKO/“allele”). b, Multiplexed PCR analysis of TgCDPK1. c, Immunofluorescence analysis of the cKO +/−ATc; endogenous MIC2 (green), HA9-tag (red) and DNA (blue). d, Immunoblot of HA9-tagged regulatable and c-Myc-tagged constitutive TgCDPK1 in cKO and complemented strains +/− Atc. Aldolase, loading control. e, Plaque formation on fibroblast monolayers, +/− ATc for 7 days.

Figure 2. TgCDPK1 is required for phenotypes associated with microneme secretion

a, Types of gliding motility as quantified by video microscopy. Student’s t test; *P < 0.05, mean ± s.e.m., N = 4 experiments. b, Invasion of fibroblasts by WT, cKO and complemented strains. Extracellular and intracellular parasites were differentially stained and enumerated as described in supplementary materials. Student’s t test; ***P<0.0005, **P<0.005, mean ± s.e.m., N = 3 experiments. c, Ionophore-induced egress of the cKO +/− ATc. Time stamp (min:sec) after calcium ionophore addition. See supplementary online videos.

Figure 3. Calcium-dependent microneme secretion requires TgCDPK1

a, Western blot analysis of microneme protein MIC2 secretion following induction by ethanol for 15 min. GRA1 shows constitutive secretion of dense granules. Student’s t test; **P < 0.005, mean ± s.e.m., N = 3 experiments. b, Ionophore-induced permeabilization detected by vacuolar DsRed leakage monitored by fluorescence video-microscopy of strains 90h + ATc . Time stamp (min:sec) after calcium ionophore addition. CytochalasinD added to prevent eggress. c-d, Quantification of maximal rate and timing of florescence loss from rupturing vacuoles. Mann-Whitney test; ***P < 0.0005, **P < 0.005, mean, N = 3 experiments.

Figure 4. PP1 derivatives specifically inhibit TgCDPK1 and block micronem-mediated functions

a, Alignment of the kinase sub-domain V highlighting the gatekeeper residue. b, Structures of 3-MB-PP1 and 3-BrB-PP1. c, Effect of 5μM 3-MB-PP1 on host cell invasion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. d, Effect of 5μM 3-MB-PP1 on MIC2 secretion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. e-f, Effect of PP1 derivatives on host lysis by T. gondii +/− 3-MB-PP1 (e) and 3-Br- PP1 (f). Mean ± s.e.m., N = 3 experiments.