The HCF101 protein is an important component of the cytosolic iron-sulfur synthesis pathway in Toxoplasma gondii

Abstract

Several key cellular functions depend on proteins harboring an iron-sulfur (Fe-S) cofactor. As these Fe-S proteins localize to several subcellular compartments, they require a dedicated machinery for cofactor assembly. For instance, in plants and algae there are Fe-S cluster synthesis pathways localizing to the cytosol, but also present in the mitochondrion and in the chloroplast, 2 organelles of endosymbiotic origin. Toxoplasma gondii is a plastid-bearing parasitic protist responsible for a pathology affecting humans and other warm-blooded vertebrates. We have characterized the Toxoplasma homolog of HCF101, originally identified in plants as a protein transferring Fe-S clusters to photosystem I subunits in the chloroplast. Contrarily to plants, we have shown that HCF101 does not localize to the plastid in parasites, but instead is an important component of the cytosolic Fe-S assembly (CIA) pathway which is vital for Toxoplasma. While the CIA pathway is widely conserved in eukaryotes, it is the first time the involvement of HCF101 in this pan-eukaryotic machinery is established. Moreover, as this protein is essential for parasite viability and absent from its mammalian hosts, it constitutes a novel and promising potential drug target.

Fig 1. TgHCF101 is a cytosolic protein.

(A) Evolutionary relationship of proteins of the MRP family. Eukaryotic sequences from HCF101 homologs were aligned and submitted to phylogenetic analysis with the maximum likelihood method. Scale bar represents 1 residue substitution per site. Bacterial P-loop ATPase sequences were used as an outgroup. HCF101 from A. thaliana is indicated by a green arrowhead, TgHCF101 by a yellow arrowhead and the 2 homologs present in C. velia are indicated by red arrowheads. NBP35 homologs from A. thaliana, T. gondii, and C. velia are indicated by purple, blue, and brown arrowheads, respectively. (B) Schematic representation of homologs for HCF101 in A. thaliana (AtHCF101), in T. gondii (TGGT1_318590) and in C. velia (Cvel_23131, Cvel_17212), and homologs for NBP35 in A. thaliana (AtNBP35), in T. gondii (TGGT1_280730), and in C. velia (Cvel_19821). Main domains are highlighted on the sequences; TP: predicted transit peptide, MIP18-like domain (or Domain of unknown function 59, DUF59), the ATPase domain and DUF971. (C) Strategy for generating the inducible knockdown of TgHCF101 by promoter replacement and simultaneous N-terminal tagging of the TgHCF101 protein in the TATi ΔKu80 cell line. (D) Diagnostic PCR for checking correct integration using the primers mentioned in (C), on genomic DNAs of a transgenic parasite clone and of the parental strain. (E) Immunoblot analysis of the cKD-TgHCF101 mutant and parental line showing efficient tagging and down-regulation of TgHCF101 starting at 24 h of treatment with ATc. Actin was used as a loading control. (F) Immunofluorescence assay showing a cytosolic signal for TgHCF101 protein (labeled with an anti-HA antibody), with no particular co-localization with the apicoplast (Apico, labeled with anti-PDH-E2 antibody), and total depletion of the protein after 48 h of ATc treatment. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 5 μm. The data underlying this figure can be found in S1 Table and in https://doi.org/10.6084/m9.figshare.28238195.

Fig 2. TgHCF101 is essential for parasite growth and survival.

(A) Plaque assays were carried out by infecting a monolayer of HFFs with TATi ΔKu80 or cKD-TgHCF101 cell lines for 7 days in the presence or absence of ATc. Scale bar = 2 mm. (B) Quantification of plaque area observed in (A). Results are expressed as percentage of lysed area relative to control (TATi ΔKu80 -ATc, set as 100% for reference). Values represented are mean ± SD from n = 3 independent biological replicates, ** p-value ≤0.01, Student t test. (C) Plaque assays were carried out with cKD-TgHCF101 parasites as described in (A) but for the ‘7d+7d-‘ condition, ATc was washed-out after 7 days and parasites were allowed to grow for another 7 days without ATc, while in the ‘7d+7d+’ condition, ATc treatment was maintained for 7 more days. The ‘7d –‘control was kept without ATc for 7 days of growth. Scale bar = 2mm. (D) Replication assay of parental (TATi ΔKu80) and transgenic cell lines (cKD-TgHCF101): parasites were pre-cultured for 48 h in the presence or absence of ATc and allowed to invade HFF-coated coverslips for another 24 h in the presence or absence of ATc, for a total of up to 72 h of treatment with ATc. Number of parasites per vacuole was quantified for each condition and expressed as a percentage, 200 vacuoles were counted for each condition. Values represented are mean ± SD of 3 independent biological replicates, **** p-value ≤0.0001 by two-way ANOVA with Dunnett’s multiple comparison test, showing a significant difference when comparing the TATi ΔKu80 control and cKD-TgHCF101+ATc parasites for percentage of vacuoles containing 1 or 8 parasites. (E) Immunofluorescence assay of cKD-TgHCF101 parasites showing asynchronous division of parasites growing in the same vacuole (outlined with a yellow dotted line) upon TgHCF101 depletion (+ATc condition: parasites pre-incubated for 48 h with ATc and allowed to invade coverslips for another 48 h in the presence of ATc). Individual parasites and budding daughter cells are outlined by anti-IMC3 antibody staining (magenta), DNA was stained with DAPI. Scale bar = 5 μm. (F) Electron microscopy of cKD-TgHCF101 parasites pre-incubated with ATc for 24 h before being released from their host cell and allowed to reinvade for 24 h in the presence (+ATc) of ATc, or grown in absence of ATc (-ATc). Inset shows magnification of a selected region. D: daughter bud, R: rhoptry, A: apicoplast, M: mitochondrion, CC: cytoplasmic cleft. The asterisk denotes unincorporated nuclear material. Scale bar = 2 μm (0.5 μm for inset magnification). (G) DNA content analysis by flow cytometry on TATi ΔKu80 and cKD-TgHCF101 parasites treated or not with ATc up to 4 days and stained with propidium iodide. 1N, 2N, 3N represent the ploidy, with <1N corresponding to parasites with less than 1 full nuclear DNA content. The data underlying this figure can be found in S1 Data and http://flowrepository.org/id/FR-FCM-Z8SR.

Fig 3. TgHCF101 depletion induces lipid droplet accumulation.

(A) Electron microscopy of cKD-TgHCF101 parasites pre-incubated with ATc for 48 h and allowed to reinvade for 24 h in the presence of ATc. Scale bar = 1 μm. The panel on the right corresponds to a magnification of the selection on the left panel, highlighting lipid droplets. Scale bar = 500 nm. (B) Fluorescent imaging of parasites from the cKD-TgHCF101 cell line treated for 72 h with ATc or in grown the absence of ATc, shows an accumulation of lipid droplets upon TgHCF101 depletion. LDs were detected with Nile red (orange), parasites are outlined with an anti-IMC3 antibody (green) and DNA is stained with DAPI. DIC = Differential interference contrast. Scale bar = 5 μm. (C, D) Correspond to the quantification of lipid droplet area and number, respectively, and 100 parasites were analyzed per condition. The parental (TATi ΔKu80) and transgenic parasites (cKD HA-TgHCF101, cKD TgABCB7, cKD TgNAR1) were grown in absence of ATc, or in presence of ATc for 72 h. Values are represented as the mean ± SD of n = 3 independent biological replicates (different symbols represent different series); ns, not significant (p-value >0.05), * p-value ≤0.05, ** p-value ≤0.01, *** p-value ≤0.001, **** p-value ≤0.0001, Student’s t test. The data underlying this figure can be found in S1 Data. cKD, conditional knock-down; LD, lipid droplet; SD, standard deviation.

Fig 4. A C-terminal TCR-like motif is necessary for TgHCF101 function.

(A) Alignment of the C-terminal region of HCF101 homologs from different eukaryotes, including the plant A. thaliana, the 2 isoforms of Apicomplexa-relative photosynthetic algae C. velia, as well as several Apicomplexa species (T. gondii, Eimeria tenella, P. falciparum, and apicoplast-less Cryptosporidium parvum). The tryptophan-containing TCR signal is highlighted in yellow. Consensus tree was obtained with protein alignment and bootstrap values (500 replicates) are indicated at the base of the nodes. (B) Schematic representation of the strategy for generating cell lines expressing myc-tagged wild-type (WT) or TCR motif-deleted (-LEW) copies of TgHCF101 by integrating an extra copy of the gene of interest by double homologous recombination at the Uracil Phosphoribosyltransferase (UPRT) locus. Negative selection with 5-fluorodeoxyuridine (FUDR) was used to select transgenic parasites based on their absence of UPRT expression. (C) Diagnostic PCR for verifying integration at the UPRT locus thanks to the primers described in B. (D) Immunoblot analysis showing expression of the additional myc-tagged copies upon depletion of the ATc-regulated HA-tagged copy. (E) Immunofluorescence with anti-myc antibody confirms the cytoplasmic localization of the extra copies. Parasite shape is outlined. DNA was stained with DAPI. Scale bar = 5 μm. (F) Plaque assay showing restoration of growth by the additional WT copy upon depletion of the ATc-regulated copy, contrarily to the TCR mutant. (G) Representative images of vacuoles (outlined) containing parasites grown continuously in the presence of ATc for 72 h and stained by Nile red (orange) for lipid droplets. DIC: differential interference contrast. DNA was stained with DAPI. Scale bar = 10 μm. (H, I) Correspond to the quantification of lipid droplet area and number, respectively; 100 parasites were analyzed per condition. WT or TCR-depleted (-LEW) parasites were grown in presence of ATc for 72 h. Values are represented as the mean ± SD of n = 3 independent biological replicates (different symbols represent different series); ns, not significant (p-value >0.05), ** p-value ≤0.01, **** p-value ≤0.0001, Student’s t test. The data underlying this figure can be found in S1 Data. HA, hemagglutinin; SD, standard deviation; TCR, targeting complex recognition.

Fig 5. TgHCF101-depleted parasites express bradyzoite-specific markers but are unable to complete their conversion.

(A) Volcano plot showing differential expression of proteins impacted by TgHCF101 depletion after 72 h of ATc treatment analyzed by label-free quantitative proteomic. X-axis corresponds to the log₂ of the fold-change (FC) and the Y-axis corresponds to the -log₁₀ of the p-value, when comparing cKD-TgHCF101 expression values to the TATi ΔKu80 parental cell line. Statistical analyses were performed with ANOVA on 4 independent biological replicates. Cut-offs were set at ≤1.5- or ≥1.5-FC and p-value ≤0.05. Significant hits corresponding to stage-specific protein are highlighted in green on the graph. (B) Clustering of bradyzoite (Bz) or tachyzoite (Tz)-specific proteins of the SRS family shows specific enrichment of bradyzoite proteins upon TgHCF101 depletion. (C) Immunofluorescence assay of cKD-TgHCF101 treated for 24 h or 48 h with ATc, cyst wall is labeled with DBL, parasites periphery is outlined with anti-IMC3 antibody and DNA is stained with DAPI. Scale bar = 10μm. (D) Corresponds to the quantification of the percentage of vacuoles presenting a DBL positive signal as shown in (C). Values are represented as the mean ± SD of n = 3 independent biological replicates, * p-value ≤0.05, Student’s t test. (E) Immunofluorescence assay of cKD-TgHCF101 treated for 7 days in the presence of ATc, cyst wall is labeled with DBL, parasites are outlined with anti-IMC3 antibody and DNA is stained with DAPI. The percentage of DBL positive vacuoles of corresponding size (more than 2 parasites per vacuole, top; or 2 parasites per vacuole or less, bottom) is specified as mean ± SD of n = 3 independent biological replicates. DIC = Differential interference contrast. Scale bar = 10 μm. The data underlying this figure can be found in S2 and S3 Tables and in S1 Data. cKD, conditional knock-down; SD, standard deviation.

Fig 6. TgHCF101 is involved in Fe-S cluster biogenesis of the CIA pathway.

(A) Volcano plot showing differential expression of proteins impacted by TgHCF101 depletion after 72 h of ATc treatment analyzed by label-free quantitative proteomic. Cut-offs were set at ≤1.5- or ≥1.5-fold change (FC) and p-value ≤0.05. Significant hits corresponding to predicted Fe-S cluster proteins are highlighted in red on the graph. (B) Immunofluorescence assay for the detection of myc-tagged TgABCE1 in the cKD TgHCF101 genetic background. Parasites were pre-incubated for 48 h in the presence of ATc and allowed to invade HFF-coated coverslips for another 48 h in the presence of ATc. The control (no ATc) was infected 24 h prior to fixation. TgABCE1 was detected with an anti-myc antibody and DNA was stained with DAPI. Parasites periphery is outlined by white dotted lines. Scale bar = 5 μm. (C) Immunoblot analysis of TgABCE1 abundance shows decrease upon TgHCF101 depletion following up to 4 days of treatment with ATc of the cKD HA-TgHCF101 TgABCE1-myc cell line. TgABCE1 was detected with anti-myc antibody and anti-actin antibody was used as a loading control. (D) Decrease of TgABCE1 expression upon TgHCF101 depletion was quantified by band densitometry analysis and normalized on the loading control of each respective lane. The relative abundance of TgABCE1 is presented as a percentage relative to the untreated control, set as 100%, for each biological replicate. Values are represented as the mean ± SD from n = 4 independent biological replicates, ** p-value ≤0.01, **** p-value ≤0.0001; ns, not significant (p-value ≥0.05), Student’s t test. (E) Immunofluorescence assay for the detection of HA-tagged TgPOLD1 in the cKD TgHCF101 genetic background. Parasites were pre-incubated for up to 4 days in presence of ATc on HFF-coated coverslips. The control (no ATc) was infected 24 h prior to fixation. TgPOLD1 was detected with an anti-HA antibody and DNA was stained with DAPI. Parasites periphery is outlined by white dotted lines. Scale bar = 5 μm. (F) Immunoblot analysis of TgPOLD1 abundance in conditions of TgHCF101 depletion following up to 4 days of treatment with ATc of the cKD TgHCF101 TgPOLD1-HA cell line. TgPOLD1 expression was detected with anti-HA antibody and anti-actin antibody was used as a loading control. (G) Changes in TgPOLD1 expression upon TgHCF101 depletion was quantified by band densitometry analysis and normalized on the loading control of each respective lane. The relative abundance of TgPOLD1 is presented as a percentage relative to the untreated control, set as 100%, for each biological replicate. Values are represented as the mean ± SD from n = 5 independent biological replicates, * p-value ≤0.05; ** p-value ≤0.01; ns, not significant (p-value ≥0.05), Student’s t test. The data underlying this figure can be found in S2 and S3 Tables and in S1 Data. cKD, conditional knock-down; CIA, cytosolic iron–sulfur cluster assembly; HA, hemagglutinin; HFF, human foreskin fibroblast.

Fig 7. TgHCF101 is associated to the CIA targeting complex and specifically interacts with TgABCE1.

(A) Volcano plot showing differential expression of TgHCF101 and co-immunoprecipitated proteins in the cKD HA-TgHCF101 cell line after TgHCF101 depletion (with 72 h of ATc treatment) or not, as analyzed by quantitative proteomic. Cut-offs were set at ≤1.5- or ≥1.5-fold change and p-value ≤0.05. Up-regulated proteins are highlighted in blue, significant hits corresponding to predicted proteins of the CIA targeting complex and target protein TgABCE1 were annotated on the graph. (B) Immunoblot analysis of a reverse co-immunoprecipitation assay of the myc-tagged TgCIA1 protein in the cKD HA-TgHCF101 background shows specific co-immunoprecipitation of TgHCF101, which is absent upon depletion by ATc. The anti-SAG1 antibody was used as a control for unspecifically bound proteins. (C) Immunoblot analysis of a reverse co-immunoprecipitation assay of the myc-tagged TgABCE1 protein showing TgHCF101 is co-immunoprecipitating. The anti-SAG1 antibody was used as a control for unspecifically bound proteins. (D) TgHCF101 interacts with TgABCE1 in a Gal4-based yeast two-hybrid assay. YRG2 cells co-transformed with AD- and BD-fusion proteins were grown to stationary phase, then serially diluted to OD₆₀₀ values ranging from 1 to 5 × 10⁻³ before being spotted onto a control plate (+HIS) to assess cell viability, and a yeast two-hybrid test plate (-HIS) to assess interaction. Plates were incubated at 30°C, and yeast growth was recorded after 5 days. TgHCF101 exhibited a strong interaction with TgABCE1, independent of cloning orientation. The strongest interaction was observed between AD-TgHCF101 and BD-TgABCE1, with co-transformed cells growing at the lowest dilution tested in the presence of 10 mM 3-aminotriazole (3AT) as a competitive inhibitor. Neither TgHCF101 nor TgABCE1 alone showed HIS3 transactivation in the presence of 3AT. These results are representative of 3 independent experiments. (E) Immunoblot analysis of puromycin incorporation in the parental (TATi ΔKu80) and cKD TgHCF101 cell lines untreated or treated with ATc for 72 h. TATi ΔKu80 treated with cycloheximide (CHX) was used as a control for translation inhibition. The puromycin signal was detected with anti-puromycin antibody, and total protein content was visualized by stain-Free imaging technology. Anti-actin antibody was also used as a loading control. (F) Variation of puromycin incorporation in the different conditions was quantified by band densitometry and normalized on the total protein content of each respective lane. Puromycin labeling is presented as a percentage relative to the untreated control, set as 100% for each biological replicate. Values are represented as the mean and SD of 4 independent biological replicates; * p-value ≤0.05; ** p-value ≤0.01; ns, not significant (p-value ≥0.05), Student’s t test. The data underlying this figure can be found in S4 Table and in S1 Data. cKD, conditional knock-down; CIA, cytosolic iron–sulfur cluster assembly; HA, hemagglutinin.

Fig 8. Schematic representation of the putative organization of the CIA pathway in T. gondii.

This scheme places TgHCF101 as an Fe-S transfer protein from the CTC to client protein ABCE1. Plant nomenclature was used for the purpose of the figure, but names for human homologs are mentioned between brackets when appropriate. CIA, cytosolic iron–sulfur cluster assembly; CTC, CIA targeting complex.