Functional Analysis of the Expanded Phosphodiesterase Gene Family in Toxoplasma gondii Tachyzoites

Abstract

Toxoplasma motility is both activated and suppressed by 3',5'-cyclic nucleotide signaling. Cyclic GMP (cGMP) signaling through Toxoplasma gondii protein kinase G (TgPKG) activates motility, whereas cyclic AMP (cAMP) signaling through TgPKAc1 inhibits motility. Despite their importance, it remains unclear how cGMP and cAMP levels are maintained in Toxoplasma. Phosphodiesterases (PDEs) are known to inactivate cyclic nucleotides and are highly expanded in the Toxoplasma genome. Here, we analyzed the expression and function of the 18-member TgPDE family in tachyzoites, the virulent life stage of Toxoplasma. We detected the expression of 11 of 18 TgPDEs, confirming prior expression studies. A knockdown screen of the TgPDE family revealed four TgPDEs that contribute to lytic Toxoplasma growth (TgPDE1, TgPDE2, TgPDE5, and TgPDE9). Depletion of TgPDE1 or TgPDE2 caused severe growth defects, prompting further investigation. While TgPDE1 was important for extracellular motility, TgPDE2 was important for host cell invasion, parasite replication, host cell egress, and extracellular motility. TgPDE1 displayed a plasma membrane/cytomembranous distribution, whereas TgPDE2 displayed an endoplasmic reticulum/cytomembranous distribution. Biochemical analysis of TgPDE1 and TgPDE2 purified from Toxoplasma lysates revealed that TgPDE1 hydrolyzes both cGMP and cAMP, whereas TgPDE2 was cAMP specific. Interactome studies of TgPDE1 and TgPDE2 indicated that they do not physically interact with each other or other TgPDEs but may be regulated by kinases and proteases. Our studies have identified TgPDE1 and TgPDE2 as central regulators of tachyzoite cyclic nucleotide levels and enable future studies aimed at determining how these enzymes are regulated and cooperate to control Toxoplasma motility and growth. IMPORTANCE Apicomplexan parasites require motility to actively infect host cells and cause disease. Cyclic nucleotide signaling governs apicomplexan motility, but it is unclear how cyclic nucleotide levels are maintained in these parasites. In search of novel regulators of cyclic nucleotides in the model apicomplexan Toxoplasma, we identified and characterized two catalytically active phosphodiesterases, TgPDE1 and TgPDE2, that are important for Toxoplasma's virulent tachyzoite life cycle. Enzymes that generate, sense, or degrade cyclic nucleotides make attractive targets for therapies aimed at paralyzing and killing apicomplexan parasites.

Keywords: Toxoplasma gondii; apicomplexan parasites; cyclic nucleotides.

FIG 1

Predicted protein domain architecture of the TgPDE family. Transmembrane domains and conserved protein domains of the 18 putative 3′,5′-cyclic nucleotide phosphodiesterases in T. gondii based on TOPCONS and NCBI Conserved Domain Database searches.

FIG 2

Creation of conditional knockdown lines for all 18 TgPDEs. (A) CRISPR-Cas9 genome editing strategy used to append an mAID-3HA, HXGPRT cassette (with 40-bp homology flanks) to the 3′ end of each TgPDE gene in the RH TIR1-3FLAG background. (B) Diagnostic PCR confirmation of successful mAID-3HA, HXGPRT integration for each TgPDE gene. Differential genomic positions of PCR1 and PCR2 are shown in panel A. WT, wild-type parental line RH TIR1-3FLAG. Tag, specific RH TgPDE-mAID-3HA clone.

FIG 3

Expression and depletion of TgPDE-mAID-3HA fusions in tachyzoites. (A) IF microscopy of intracellular RH TgPDE-mAID-3HA parasites labeled with mouse anti-HA and goat anti-mouse IgG Alexa Fluor 488. Bar, 5 μm. (B) Immunoblots of lysates from RH TIR1-3FLAG (TIR1) and RH TgPDE-mAID-3HA (TgPDE) parasites treated with vehicle (EtOH) or 0.5 mM IAA for 18 h. Blots were probed with mouse anti-HA, rabbit anti-SAG1, goat anti-mouse-AFP800, and goat anti-rabbit-AFP680. The table presents the predicted total mass of each TgPDE, including the mAID-3HA tag (12 kDa). Arrows indicate immunoblot-detectable TgPDE-mAID-3HA fusions in tachyzoites.

FIG 4

Contribution of each TgPDE to tachyzoite growth. (Inset) Auxin-inducible protein degradation. Plaques formed on HFF monolayers by RH TIR1-3FLAG (TIR1) or derivative RH TgPDE-mAID-3HA lines treated with vehicle (EtOH) or 0.5 mM IAA for 8 days. Bar = 5 mm. Plaque count data are means and standard deviations (SD) (n = 6 or 9 replicates, combined from 2 or 3 trials, respectively). For baseline consistency, plaque counts were normalized to 100 vehicle-treated plaques for each line. Plaque area data are means and SD (n = 10) from the representative images shown. Unpaired Student's t test (ethanol versus IAA) was used. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant.

FIG 5

Phosphodiesterase activity of immunoprecipitated TgPDE1 and TgPDE2. (A) Immunoprecipitation strategy for purifying tagged TgPDE1 and TgPDE2 proteins from tachyzoite native lysates. Untagged parental line RH TIR1-3FLAG (TIR1) served as a negative control. (B) Representative immunoblot of immunoprecipitation fractions probed with rat anti-HA and goat anti-rat IRDye 800CW. T, total lysate; S, soluble lysate; E, eluate. (C) Cyclic AMP phosphodiesterase activity of immunoprecipitated elution fractions incubated 1:1 with 0.2 μM cAMP for 2 h at 37°C. Standards shown were incubated 1:1 with PDE storage buffer for 2 h at 37°C. (D) Cyclic GMP phosphodiesterase activity of immunoprecipitated elution fractions incubated 1:1 with 20 μM cGMP for 2 h at 37°C. Standards shown were incubated 1:1 with PDE storage buffer for 2 h at 37°C. (C and D) Mean and SD (n = 3) from one of four trials (n = 4) with similar outcomes. Statistical significance was determined using an unpaired Student's t test (TIR1 versus TgPDE1; TIR1 versus TgPDE2). ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant.

FIG 6

Role of TgPDE1 and TgPDE2 in the tachyzoite lytic cycle. RH TIR1-3FLAG (TIR1) and derivative RH PDE-mAID-3HA parasites (TgPDE1; TgPDE2) were analyzed for defects in the lytic cycle following conditional knockdown of TgPDE1 or TgPDE2 with auxin. (A to C) Replication assay of parasites grown in HFFs treated with vehicle (EtOH) or 0.5 mM IAA for 22 h from 3 independent trials. (A) IF microscopy of fixed parasites labeled with antibodies to TgSAG1, TgGRA7, and the DNA dye Hoechst 33258. Bar = 5 μm. Irregular vacuoles contain an irregular number of parasites or parasites with abnormal morphology. (B) Distribution of parasites per vacuole, with SD. For each line, percentages of parasites in each bin were compared between EtOH and IAA treatment and statistical significance was determined using a two-way ANOVA with Tukey’s multiple-comparison test. ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. (C) Mean parasites per vacuole and SD. Unpaired Student's t test (EtOH versus IAA) was used. **, P ≤ 0.01. (D) Parasite invasion of HFFs (20 min) following treatment with EtOH or 0.5 mM IAA for 16 h from two or three independent trials. Shown are the mean numbers of attached and invaded parasites per field, with SD, as determined by IF microscopy using differential permeabilization and immunolabeling with rat and rabbit TgSAG1 antibodies. Bar = 5 μm. Unpaired Student's t test (EtOH versus IAA) was used. *, P ≤ 0.05. (E) Parasite egress from HFFs (40 h) following treatment with EtOH of 0.5 mM IAA for 22 h and stimulation with vehicle (DMSO), 0.5 mM zaprinast, or 2 μM A23187 for 5 min from 3 independent trials. Shown are the mean percentages of intact and egressed vacuoles with standard errors of the means (SEM) based on IF microscopy using immunolabeling with antibodies against TgSAG1 and TgGRA7. Bar = 100 μm. Two-way ANOVA with Tukey’s multiple-comparison test was used. *, P ≤ 0.05; ****, P ≤ 0.0001. (F) Parasite motility on a BSA-coated cover glass in EC buffer following 14 h treatment with EtOH or 0.5 mM IAA. Following a 20-min incubation, parasites were fixed to the cover glass and immunolabeled with TgSAG1 antibodies to detect the parasites and their motility trails by IF microscopy. Shown are mean percentages of parasites and SD with no trail, a short trail (<1 body length), or a long trail (>1 body length) from one of two independent trials. Bar = 30 μm. Two-way ANOVA with Tukey’s multiple-comparison test was used. *, P ≤ 0.05; **, P ≤ 0.01. (G) Microneme secretion assay following 14 h treatment with EtOH or 0.5 mM IAA and stimulation with DMSO or 0.5 mM zaprinast in EC buffer for 10 min as detected by immunoblotting with TgMIC2 and TgGRA7 (constitutively secreted control protein) antibodies. Representative immunoblots are shown. P, 10% of unstimulated parasite pellet lysate; M, mock-stimulated secreted fraction; Z, zaprinast-stimulated secreted fraction.