Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3

Abstract

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

Figure 1

ApiAT5‐3 localises to the plasma membrane and is phosphorylated at serine 56 upon ionophore treatment. A. Quantification of the phosphorylation state of residues in the ApiAT5‐3 N‐terminus in TgCDPK3 KOs and during ionophore‐induced egress (from Treeck et al., 2014). Upper panel: The heatmap shows differential phosphorylation of S56 in TgCDPK3 mutants compared to WT parasites, but not any other of the identified phosphorylation sites. Intracellular (IC) and extracellular (EC) parasites with and without 1 µM ionophore (iono). P‐site = phosphorylation site. Numbers represent residue position. Black ‘x’ = phosphorylation site not identified. Fold changes are log². Bottom panel: Change in relative phosphorylation of ApiAT5‐3 and proteins with previously described ionophore‐dependent phosphorylation sites, measured after addition of 8 µM ionophore over 60 s. Numbers after the identifier represent the phosphorylation site quantified. B. ApiAT5‐3 was detected by Western blot analysis of ApiAT5‐3::HA cell lysate using an anti‐HA antibody. Loading control anti‐Toxo. C. IFA of ApiAT5‐3::HA expressing parasites shows that ApiAT5‐3 localises to the periphery of the intracellular tachyzoite. Red = HA, Green = SAG1, Blue = DAPI. Scale bar 5 µm.

Figure 2

ApiAT5‐3 is essential for parasite proliferation. A. Generation of the ApiAT5‐3_loxP line using CRIPSPR/Cas9 to increase site‐directed integration. Protospacer adjacent motif (PAM) indicated by black arrows. Primer pairs represented by coloured triangles. B. Left panel: PCR analysis shows correct integration of the ApiAT5‐3_loxP construct at both the 3ʹ and 5ʹ ends and a loss of WT apiAT5‐3 at the endogenous locus. White * = non‐specific bands. Right panel: Addition of RAP leads to correct recombination of the loxP sites. C. Fluorescent microscopy of ApiAT5‐3_loxP parasites 24 h after addition of DMSO or RAP. Scale bar 5 µm. D. Plaque assay showing loss of plaquing capacity of ApiAT5‐3_loxP parasites upon RAP treatment. E. Parasite per vacuole number shown as mean %, n = 3. F. Stills from live video microscopy at 36, 42 and 45 h into third lytic cycle post‐RAP treatment. Red = RH Tom, dashed white line = intact WT ApiAT5‐3_loxP vacuoles, green = apiAT5‐3 KO. Scale bar 20 µm. G. IIE assay showing no significant difference between DMSO‐ and RAP‐treated ApiAT5‐3_loxP (at 30 h into lytic cycle 2 post‐DMSO/RAP treatment). Statistical analysis using multiple comparison two‐way ANOVA, n = 2.

Figure 3

∆apiAT5‐3 parasites display a transcriptional response related to amino acid starvation. A. Extracted reads for recodonised apiAT5‐3 from RNA sequence data show a significant reduction of apiAT5‐3 transcripts in RAP‐treated ApiAT5‐3_loxP lines 60 h post‐RAP treatment compared to RAP‐treated ApiAT5‐3_loxP^dDiCre parasites. B. Heatmap of genes that change significantly (adjusted p < 0.05) in transcript read number between WT and ∆apiAT5‐3 60 h post‐addition of RAP. C. Gene ontology term enrichment shows that genes involved in translation processes are significantly enriched among the differentially expressed genes 60 h post‐RAP treatment.

Figure 4

∆apiAT5‐3 ^{ApiAT5‐3_S56A} demonstrates a fitness defect. A. Generation of the ApiAT5‐3^{ApiAT5‐3/_S56A/_S56D} complementation lines. PAM indicated by black arrow. Primer pairs represented by coloured triangles. B. PCR analysis shows correct integration of the ApiAT5‐3_loxP construct at both the 3ʹ and 5ʹ ends and a loss of uprt. White * = nonspecific band. C. IFA of ApiAT5‐3^{ApiAT5‐3/_S56A/_S56D::HA} expressing parasites shows that ApiAT5‐3 is correctly trafficked to the periphery of the intracellular tachyzoite in both the presence (DMSO) and absence (RAP) of the endogenous apiAT5‐3. Red = HA. Green = YFP, indicating correct excision of the endogenous apiAT5‐3. Scale bar 10 µm. D. Geometric mean of red fluorescence calculated by flow cytometric analysis of complemented parasites, probed with red fluorescent anti‐HA antibody. Statistical analysis carried out using multiple comparison, two‐way ANOVA, ns = not significant. All complemented lines differ significantly in mean fluorescence from ApiAT5‐3_loxP (p < 0.0001), n = 3. E. Growth competition assay by flow cytometry shows that ∆apiAT5‐3 ^{ApiAT5‐3_S56A} parasite growth is reduced relative to the non‐excised ApiAT5‐3^{ApiAT5‐3_S56A} line. Statistical analysis using multiple comparison, two‐way ANOVA of mean ratio to day 0 normalised to 1. ***p < 0.001, n = 3.

Figure 5

Functional analysis of the ApiAT5‐3 transporter. A. X. laevis oocytes expressing ApiAT5‐3 demonstrate a significant increase in ¹⁴C‐L‐tyrosine uptake. Ten oocytes per experiment. Analysis carried out using a two‐tailed, paired, Student’s t‐test. ***p < 0.001 Box plots show mean, first and third quartile and SD, n = 5. B. Extracellular, RAP‐treated, ApiAT5‐3_loxP tachyzoites, labelled with ¹³C‐L‐tyrosine or ¹³C‐L‐isoleucine, display a marked decrease in tyrosine but not isoleucine import, relative to WT, n = 2. C. Plaque assay shows no rescue of growth of RAP‐treated ApiAT5‐3_loxP on addition of excess (2 mM) L‐tyrosine.