TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells

Abstract

The phylum Apicomplexa comprises a group of obligate intracellular parasites of broad medical and agricultural significance, including Toxoplasma gondii and the malaria-causing Plasmodium spp. Key to their parasitic lifestyle is the need to egress from an infected cell, actively move through tissue, and reinvade another cell, thus perpetuating infection. Ca(2+)-mediated signaling events modulate key steps required for host cell egress, invasion and motility, including secretion of microneme organelles and activation of the force-generating actomyosin-based motor. Here we show that a plant-like Calcium-Dependent Protein Kinase (CDPK) in T. gondii, TgCDPK3, which localizes to the inner side of the plasma membrane, is not essential to the parasite but is required for optimal in vitro growth. We demonstrate that TgCDPK3, the orthologue of Plasmodium PfCDPK1, regulates Ca(2+) ionophore- and DTT-induced host cell egress, but not motility or invasion. Furthermore, we show that targeting to the inner side of the plasma membrane by dual acylation is required for its activity. Interestingly, TgCDPK3 regulates microneme secretion when parasites are intracellular but not extracellular. Indeed, the requirement for TgCDPK3 is most likely determined by the high K(+) concentration of the host cell. Our results therefore suggest that TgCDPK3's role differs from that previously hypothesized, and rather support a model where this kinase plays a role in rapidly responding to Ca(2+) signaling in specific ionic environments to upregulate multiple processes required for gliding motility.

Figure 1. TgCDPK3 localizes to the plasma membrane through a putative N-terminal acylation motif.

A) Tagging of the tgcpkd3 endogenous locus with 3HA. TgCDPK3-3HA runs at the expected size by western blot, showing a size shift corresponding to the addition of the 3HA epitope by probing with αTgCDPK3. TgCDPK3-3HA shows clear banding using αHA, with no band seen in wild-type (wt(ΔKu80)) parasites. B) Staining TgCDPK3-3HA parasites with αHA and αTgGAP45 shows co-localization between TgCDPK3 and TgGAP45 at the parasite periphery, likely through plasma membrane targeting as judged by staining of the parasite residual body (white arrow). C) Substitution mutations of the putative acylated residues Gly2 and Cys3 in full-length ectopic copies of TgCDPK3 disrupts its peripheral targeting, showing these residues are necessary for its localization. D) The 15 most N-terminal amino acids of TgCDPK3 impart plasma membrane localization to mOrange fluorescent protein, but mutations in the putative acylated residues identical to those described above disrupt this pattern. White arrow = concentration of TgCDPK3_NC3A at the parasite periphery.

Figure 2. Knockout of TgCDPK3 by double homologous recombination.

Wild-type (ΔKu80) = wild-type parasites. A) Design of ΔCDPK3-TOXOY72 cosmid construct, whereby double homologous recombination replaces the 3′ half of the genomic locus of TgCDPK3 with a CAT resistance cassette. Bold line shows TOXOY72 cosmid backbone. Schematic also shows hybridization site for Southern blot probe (“Pr”), recognition site for αTgCDPK3 antibody (“Ab”), and restriction sites used in Southern blotting (K = KpnI, M = MluI, S = SacII). B) Southern blot analyses of ΔTgCDPK3. KpnI digest control shows identical banding pattern between wild-type and ΔTgCDPK3 parasites, whereas digestion of gDNA with SacII/MluI shows the expected size drop of in banding between wild-type and ΔTgCDPK3 (“*”). C) Knockout of a clonal transgenic line, ΔTgCDPK3, was confirmed with αTgCDPK3 antibody, showing lack of any significant banding by western blot compared to wild-type parasites. αTgMIC2 loading control shows equal loading of parasite lysates.

Figure 3. TgCDPK3 is required for calcium ionophore-induced egress from host cells.

A) Mixing wild-type and ΔTgCDPK3 parasites shows knockout of TgCDPK3 causes T. gondii to be inefficient in ability to complete the lytic lifecycle in vitro, being effectively out-competed by wild-type by day 18 of the experiment. B) Invasion rates of wild-type and ΔTgCDPK3 parasites shows no significant difference over a range of time points. C) Live motility assay of wild-type and ΔTgCDPK3 parasites. i. Overall proportion of motile versus immotile parasites between wild-type and ΔTgCDPK3 parasites, either with or without calcium ionophore A23187 stimulation. Both strains show amplification of motility following A23187 treatment, with no significant difference in level of amplification. ii. Proportions of twirling, helical and circular motility exhibited by wild-type and ΔTgCDPK3 parasites. ΔTgCDPK3 parasites show a slight preference for twirling motility over helical following A23187 stimulation, as compared to wild-type. D) ΔTgCDPK3 mutants show a severe defect in ability to egress from host cells following stimulation of calcium signaling with calcium ionophore A23187. E) ΔTgCDPK3 mutants show a defect in egress from host cells following stimulation of calcium signaling with reducing agent DTT. Egress levels are normalized against wild-type. F) Live time-lapse microscopy of ΔTgCDPK3 parasites show an inability to activate motility and escape host cells up to and beyond 10 min after ionophore stimulation, whereas wild-type parasites activate egress within 1.30 min. Calcium ionophore is added at 30 sec time point. G) An inability to activate egress upon ionophore stimulation is not due to a general defect in calcium signaling in ΔTgCDPK3, as mutants show extrusion of conoids coincident with the normal timing of wild-type extrusion and egress.

Figure 4. Intracellular ΔTgCDPK3 parasites are defective in calcium-stimulated microneme secretion.

A) Live fluorescent time lapse microscopy of wild-type and ΔTgCDPK3 parasites, transiently expressing an ectopic DsRed protein which is targeted to the PVM, and treated with cytD to disrupt motility. Wild-type parasites permeabilize the PVM coincident with the timing of normal egress following addition of A23187, as seen by diffusion of DsRed from the PVM through the host cell. ΔTgCDPK3 parasites cannot permeabilize the PVM, and DsRed remains within the PVM. Calcium ionophore is added at 30 sec time point. B) Following stimulation with A23187, wild-type parasites display microneme proteins TgSUB1 and TgMIC11 at the apical tip, indicative of microneme secretion, as seen by co-staining with the surface marker TgSAG1. TgGRA1 diffuses from the PVM through the host cell coincident with microneme secretion by wild-type parasites, confirming DsRed permeabilization results. Ionophore-treatment of ΔTgCDPK3 parasites stimulates no such microneme protein secretion, and TgGRA1 remains within the PVM. C) Stimulating calcium signaling in extracellular ΔTgCDPK3 parasites by treatment with either 1% EtOH or A23187 shows no defect in secretion of a range of microneme proteins in supernatant samples, as compared to wild-type parasites. “*” denotes weak banding of unprocessed TgMIC2 in ΔTgCDPK3 supernatant samples, indicating a small level of inadvertent parasite lysis, but not sufficient to explain levels of microneme proteins seen in ΔTgCDPK3 supernatant.

Figure 5. TgCDPK3 kinase activity is required for activation of gliding motility in high potassium environments.

A) Live time lapse microscopy of host cells co-infected with YFP-expressing wild-type and ΔTgCDPK3 parasites. ΔTgCDPK3 parasites are able to activate gliding motility and egress shortly after wild-type parasites escape the host cell. White arrows indicate egressing ΔTgCDPK3 parasites. B) Selective saponin permeabilization of PVM and host cell membrane induces wild-type egress if treated in a low potassium (“extracellular” – EC) buffer, and is not significantly enhanced by stimulated with calcium ionophore. ΔTgCDPK3 parasites show significantly lower levels of egress then wild-type whether ionophore-stimulated or not, but egress is enhanced with A23187 stimulation. C) Wild-type parasites are inhibited in permeabilization-induced egress in a high potassium (“intracellular” – IC) buffer, relative to EC buffer treatment, but this can be overcome by stimulation of calcium signaling by A23187. ΔTgCDPK3 parasites are severely inhibited in egress under both conditions, and are not able to overcome inhibition following ionophore stimulation. “−” indicates A23187-negative (DMSO-treated controls) , “+” indicates A23187-treated samples. “*” indicates significant difference between DMSO- and A23187-treated samples (P<0.05, two-tailed Student's t-test). Error bars = ± s.d.

Figure 6. Activity of TgCDPK3 requires putative acylated residues in the consensus N-terminal motif.

ΔTgCDPK3 parasites transfected with the wild-type (TgCDPK3_wt) or palmitoylation mutant (TgCDPK3_C3A) ectopic copies of TgCDPK3 show wild-type levels of egress, following stimulation with A23187, indicating successful complementation. Complementation with myristoylation (TgCDPK3_G2A) or double mutants (TgCDPK3_G2AC3A) is not successful. Error bars = ± s.d.