ZFT is a ZIP-domain containing protein with dynamic localisation.

a. Alignment of ZIP proteins from T. gondii (TGME49_261720), T. gondii (TGME49_266800), P. falciparum (PF3D7_1022300), HsZIP1 and HsZIP2. Key conserved residues in the binuclear metal centres (BNC) M1 and M2 in transmembrane domains a4 and a5, which have been shown to be required for metal binding are highlighted. Conserved glutamate residues highlighted in purple. b. Alphafold model of TGME49_261720 dimer with key residues highlighted c. Schematic of F3ΔHX ZFT-3HAzft strain construction to endogenously tag ZFT with 3xHA epitope tags at the C-terminal under the endogenous promoter with the endogenous 3’ UTR. d. Western blot analysis of ZFT-3HAzft confirming the expected band size (50 kDa). Non-specific bands indicated with asterisks. CDPK1 (TGME49_301440) used as a loading control. e. Immunofluorescence of ZFT-3HAzft demonstrating dynamic peripheral or basal localisation. Scale bars 5 μm. f. Percentage of ZFT-3HAzft localisation (peripheral or basal) in respect to number of parasites per vacuole. 100 parasites were counted for each condition. Bars at mean of four independent experiments, ± SD. g. Mean fluorescence intensity (MFI) of ZFT-3HAzft per parasite in relation to the number of parasites per vacuoles. Each point represents individual MFI/parasite values from three independent experiments. Bars at mean of the experiments ± SD. p values from ordinary one-way ANOVA, Tukey corrected for multiple comparisons.

ZFT-3HAzft is regulated by iron, and overexpressing ZFT leads to a change in iron sensitivity.

a. Western blot analysis showing ZFT-3HAzft expression levels 18 h post infection in untreated and high iron (500 μM FAC) conditions. CDPK1 was used as a loading control. b. Quantification of ZFT-3HAzft from three independent experiments. Bars at mean ± SD. p values from two-tailed paired t test. c. As above, at 24 h post infection in untreated and high iron (500 μM FAC) conditions. d. Quantification of ZFT-3HAzft from three independent experiments. Bars at mean ± SD. p values from two-tailed paired t test. e. Immunofluorescence assay of ZFT-3HAzft at 24 h post infection in untreated and high iron (500 μM FAC) conditions. Scale bars 5 μm. f. Quantification of ZFT-3HAzft localisation (peripheral/basal or no signal) in untreated and high iron conditions. Bars at mean of three independent experiments, ± SD. g. Schematic of ZFT-Ty strain construction to overexpress ZFT from the TUB8 promoter in RHtdTomato parental line. h. Western blot of ZFT-Ty showing expected size. i. Plaque assay of parental and ZFT-Ty parasites in untreated conditions. Quantification of plaque number (j) and area (k) for parental and ZFT-Ty parasites, p values are unpaired two-tailed t test. Points represent individual plaque areas from three independent experiments, bars at mean ± SD. p values from two-tailed unpaired t test with Welch’s correction. Graphs showing mean parasite EC50 for FAC (l) and DFO (m) for RHtdTomato and ZFT-Ty parasites. Each point represents an independent experiment, bars at mean of n=3, ± SD. p values from two-tailed unpaired t test.

ZFT knockdown blocks parasite replication.

a. Schematic of F3ΔHX T7S4-ZFT under the T7S4 inducible promoter. b. RT-qPCR showing ZFT mRNA abundance, relative to 0 h, normalised to actin. Bars at mean of three independent experiments, ± SD. p values from two-tailed unpaired t test. c. Schematic of F3ΔHX T7S4-ZFT-3HAsag1 under the T7S4 inducible promoter. d. Western blot demonstrating successful knockdown of ZFT at 24, 48, 72 and 96 h post ATc induction. CDPK1 used as a loading control. e. Quantification of (d), relative to 0 h, normalised to CDPK1. Bars at mean of four independent experiments, ± SD. p values from two-way ANOVA, Tukey corrected for multiple comparisons. f. Immunofluorescence assay showing T7S4-ZFT-3HAsag1 expression in untreated parasites and after induction with ATc for 48 h. Scale bar 5 μm. g. Blue native PAGE of T7S4-ZFT-3HAsag1, untreated and + ATc 48 h induction, showing a ZFT-HA complex at around 100 kDa. CDPK1 used as a loading control. h. Plaque assay of parental and T7S4-ZFT parasites untreated and ATc treated, 6 days, showing knockdown of ZFT renders the parasite unable to form plaques in the host cell monolayer. i. Quantification of (h) showing number of plaques, normalised to parental untreated. Bars at mean of three independent experiments, ± SD. p values from two-way ANOVA, Tukey corrected for multiple comparisons. j. Quantification of plaques area from (h). Points represent individual plaques from three independent experiments. Bars at mean ± SD. p values from ordinary one-way ANOVA, Tukey corrected for multiple comparisons. k. Plaque assays with addition of 200 μM FAC does not rescue ZFT knockdown parasites. l. Quantification of plaque area generated by T7S4-ZFT parasites in untreated and high iron (200 μM FAC) conditions. Points represent individual plaque areas from three independent experiments. Bars at mean ± SD. p values are from two-tailed unpaired t test with Welch’s correction. m. Quantification of plaque number, normalised to parental untreated. Bars at mean of three independent experiments, ± SD. p values from two-way ANOVA, Tukey corrected for multiple comparisons. n. Quantification of plaque area generated by parental and T7S4-ZFT parasites with and without ATc, in the presence of excess iron (200 μM FAC). Points represent individual plaque areas from three independent experiments. Bars at mean ± SD. p values from ordinary one-way ANOVA, Tukey corrected for multiple comparisons.

ZFT knockdown reduces mitochondrial respiration and the expression of Fe-S protein SDHB.

a. Immunofluorescence assay of T7S4-ZFT-3HAsag1 in the absence and presence of ATc for 24 h and 48 h, staining for CPN60 (apicoplast marker). b. Western blot analysis of T7S4-ZFT with and without ATc 48 h staining for lipoic acid. TgPDH-E2 and TgOGDH-E2 were detected 48 h post ZFT knockdown, suggesting the apicoplast is functional. c. Immunofluorescence assay of T7S4-ZFT parasites with and without ATc staining for CPN60 (apicoplast marker) following a further round of mechanical release and invasion to examine if ZFT knockdown has a delayed death phenotype on the apicoplast. Scale bar 5 μm. d. Quantification of % of apicoplasts from (c) with and without ATc that appeared normal or elongated. 100 vacuoles for each condition were quantified. Bars at mean of three independent replicates, ± SD. e. Quantification of the percentage of parasites per vacuole with and without ATc following a further round of mechanical release and invasion. 100 vacuoles for each condition were counted. Bars at mean of three independent replicates, ± SD. f. Immunofluorescence assay as above, staining for TOM40 (mitochondrial marker). Scale bar 5 μm g. Mitochondrial oxygen consumption rate (mOCR) of T7S4-ZFT, untreated or ATc treated for 48 h via Seahorse assay. Quantification of basal mOCR (h) and maximal mOCR (i). Bars at mean of three independent experiments, ± SD. p values are from two-tailed unpaired t test with Welch’s correction. j. Metabolic map demonstrating ZFT knockdown shits parasites to a more quiescent state. Points at mean, ± SD. k. Western blot of T7S4-ZFT SDHB-3HA, untreated and ATc treated for 48 h. CDPK1 used as a loading control. l. Quantification of SDHB-3HA expression, relative to uninduced, normalised to CDPK1. Bars at mean of three independent experiments, ± SD. p values are two-tailed paired t test. m. Complex IV activity assay of T7S4-ZFT parasites with and without ATc 48 h showing ZFT knockdown reduces complex IV activity. CDPK1 used as a loading control.

Knocking down ZFT triggers partial bradyzoite differentiation.

a. Immunofluorescence of T7S4-ZFT parasites uninduced or induced with ATc for 48 h. Highlighted is a region where parasites are disorganised and asynchronously replicating. Scale bar 5 μm. b. Quantification of the percentage of parasites/vacuole in parental and T7S4-ZFT parasites in the presence and absence of ATc for 48 h. 100 vacuoles for each condition were counted. Bars at mean of four independent replicates, ± SD. c. TEM images of T7S4-ZFT parasites with and without ATc for five days. Detail highlights regions of PVM. Yellow arrows point to enlarged vesicles in the PV space. Scale bar 500 nm. d. Immunofluorescence of T7S4-ZFT-3HAsag1 untreated and + ATc 72 h, stained with BAG1, a bradyzoite specific marker. Scale bar 5 μm. e Immunofluorescence assay of T7S4-ZFT-3HAsag1 untreated and + ATc 4 days, stained with SAG1 and DBL, a cyst wall-binding lectin. Scale bar 5 μm. Quantification of % vacuoles expressing BAG1 (f) and DBL (g). 100 vacuoles for each condition counted, bars at mean of three independent experiments, ± SD. p values from two-way ANOVA, Tukey corrected for multiple comparisons. h. Plaque assays of parental line treated with ATc for 6 days, and T7S4-ZFT parasites, treated either with ATc for 6 days, or for 3 days following washout and 6 days recovery. i. Quantification of plaque number recovery following ATc washout, normalised to parental parasites. Bars at mean of four independent experiments, ± SD. p values from two-tailed unpaired t test.

ZFT expression depends on the availability of zinc, and ZFT expression complements lack of zinc transport in S. cerevisiae.

a. Plaque assays showing that the addition of 25 μM ZnSO4, and the combination of 25 μM ZnSO4 and 200 μM FAC, cannot rescue ZFT knockdown. b. Number of plaques after treatments. Bars at mean of n=3, ± SD. p values from two-way ANOVA, Tukey corrected for multiple comparisons. c. Plaque areas of parasites treated with excess zinc (25 µM ZnSO4). Points represent individual plaque areas from two (parental -ATc) or three (all other conditions) independent experiments. Bars at mean ± SD. p values from ordinary one-way ANOVA, Tukey corrected for multiple comparisons. d. Plaque area from T7S4-ZFT parasites in untreated and high zinc (25 μM ZnSO4) conditions. Points represent individual plaque areas from three independent experiments. Bars at mean ± SD. p values are from two-tailed unpaired t test with Welch’s correction. e. Graph showing mean EC50 for zinc for parental and ZFT-Ty parasites. Bars at mean of n=3, ± SD. p values from two-tailed unpaired t test. f. Western blot analysis showing ZFT-3HAzft expression levels 18 h post infection in untreated, high zinc (50 μM ZnSO4) and 5 μM TPEN conditions. CDPK1 used as a loading control. g. Quantification of ZFT-3HAzft levels at 18 h from three independent experiments. Bars at mean ± SD. p values from one-way ANOVA, Tukey corrected for multiple comparisons. h. Western blot as above, at 24 h post infection. i. Quantification of ZFT-3HAzft levels at 24 h from three independent experiments. Bars at mean ± SD. p values from one-way ANOVA, Tukey corrected for multiple comparisons. j. Spot assay showing TgZFT expression rescues growth in Δzrt1/2 S. cerevisiae. Representative of two independent biological experiments. k. Under zinc chelation (5 mM EGTA), TgZFT allows Δzrt1/2 growth. Representative of two independent biological experiments.

Knocking down ZFT results in a decrease in parasite associated metal.

a. Live cell imaging and quantification of FerroOrange stained parental parasites, untreated or treated with 200 μM FAC for 48 h. Scale bar 5 μm. Points represent the MFI of FerroOrange/parasite from four independent experiments. Bars at mean ± SD. p value from two-tailed unpaired t test with Welch’s correction. b. Live cell imaging and quantification of FerroOrange-stained T7S4-ZFT parasites with and without ATc for 48 h. Points are MFI of FerroOrange/parasite from four independent experiments. Bars at mean ± SD. p value from two-tailed unpaired t test with Welch’s correction. c. Live cell imaging and quantification of FluoZin-3AM stained parental parasites treated with 50 μM ZnSO4 for 24 h. Points represent MFI of FluoZin-3AM/parasite from three independent experiments. Bars at mean ± SD. p value from two-tailed unpaired t test with Welch’s correction. d. Live cell imaging and quantification of FluoZin-3AM stained T7S4-ZFT parasites with and without ATc for 48 h. Scale bar 5 μm. Points represent the MFI of FluoZin-3AM per parasite from three independent experiments. Bars at mean ± SD. p value from two-tailed unpaired t test with Welch’s correction. e. ICP-MS quantification of iron/parasite (fM) from T7S4-ZFT parasites with and without ATc at 24 h and 48 h. Bars at mean (n = 7 for – ATc, n = 5 for + ATc 24 h and n = 2 for + ATc 48 h) ± SD. p values from one-way ANOVA, Tukey corrected for multiple comparisons. f. ICP-MS quantification of zinc/parasite (fM) from T7S4-ZFT parasites with and without ATc 24 h and 48 h. Bars at mean (n = 5 for – ATc and + ATc 24 h and n = 2 for + ATc 48 h) ± SD. p values from one-way ANOVA, Tukey corrected for multiple comparisons. XFM of extracellular T7S4-ZFT parasites, untreated and treated for 24 and 48 h, showing phosphorus, sulphur, iron (g) and zinc (h). i. XFM of untreated parasites showing iron and zinc colocalisation, along with phosphorus. Quantification of Fe (j) and Zn (k) following ZFT knockdown. Bars at mean ± SD. p values from one-way ANOVA, Tukey corrected for multiple comparisons. l. Quantification of changes in Fe, Zn, S and P as a percentage of untreated cells, following the addition of ATc. Bars at mean ± SD. Scale bar for all images 5 μm.

A list of the gRNA sequences used in this study.

List of primer sequences used in this study