Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase

Abstract

The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined. Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii. We identify approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth. We demonstrate that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex. We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect. Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites.

Keywords: Toxoplasma; apicomplexans; infectious disease; microbiology; mitochondria; proteomics.

Figure 1.. Biotinylation of mitochondrial matrix proteins in T.gondii parasites expressing mtAPEX and mtBirA*.T

(A–B) Immunofluorescence assays of parasites expressing c-myc-tagged, mitochondrially-targeted APEX (A) and BirA* (B), labelled with anti-c-myc (green) and the mitochondrial marker TgTom40 (red). Scale bars are 2 µm. (C–D) Western blots of parasites expressing c-myc-tagged, mitochondrially-targeted APEX (C) and BirA* (D), labelled with anti-c-myc. (E) Oregon Green-conjugated avidin (Avidin-OG) labelling of T. gondii parasites expressing mtAPEX, and cultured in the absence (top) or presence (bottom) of biotin-phenol and H₂O₂. Biotinylated proteins are labelled in green. (F) Avidin-OG labelling of T. gondii parasites expressing mtBirA*, and cultured in the absence (top) or presence (bottom) of biotin. Biotinylated proteins are labelled in green. Scale bars are 2 µm. (G) Neutravidin-HRP protein blot of WT, mtBirA* or mtAPEX parasites cultured in the presence of biotin or biotin-phenol. (H) Western blots of the mitochondrial matrix marker mtHsp60 and the mitochondrial intermembrane space marker cyt c in WT, mtBirA* or mtAPEX parasites cultured in the presence of biotin (lanes 1 – 4) or biotin-phenol (lanes 5 – 8). Parasites were either harvested following treatment to yield the total (T) protein fraction, or biotinylated proteins were purified on a streptavidin-agarose column to yield the bound (B) fraction.

Figure 1—figure supplement 1.. Map of the pBTM₃ plasmid vector, showing the AvrII, NdeI and NotI cut sites between which the APEX and BirA* cassettes were ligated (open reading frame of enzyme shown in green), the position of mitochondrial targeting leader sequence of TgHsp60 (Hsp60_L; red), the 3x c-myc tag (yellow), and the positions of the phleomycin resistance marker (Phl^R) for T.gondii selection, the ampicillin resistance marker for E. coli selection (Amp^R), and the origin of replication (Ori; all magenta).

Note: vector is not drawn to scale.

Figure 2.. The mitochondrial proteome of T.gondii.

(A–B) Volcano plots showing the log₂ protein ratios vs –log10 p values of biotinylated proteins in WT compared to mtAPEX (WT/APEX) samples (A) and in WT compared to mtBirA* samples (WT/BirA*) (B) following the quantitative pipeline analysis. Proteins were deemed to be enriched in the mitochondrion if the log₂ fold change in protein expression was ≤−2.5 and the p value ≤ 0.001 (red). (C) Venn diagram of the mtAPEX and mtBirA* proteomes. 161 proteins were identified in both proteomes, while 52 were unique to the mtAPEX proteome and 208 unique to the mtBirA* proteome. (D) Metabolic map of expected mitochondrial proteins (circles), showing proteins present (blue) and absent (yellow) from the T. gondii mitochondrial proteome. Black arrows represent the flow of metabolites through metabolic pathways in the mitochondrion, and blue arrows depict the flow of ions, minerals or metabolic pathway products.

Figure 2—figure supplement 1.. Analysis of putative mitochondrial targeting peptides in the T.gondii mitochondrial proteome.

Pie chart depicting mitochondrial targeting peptide predictions of the T. gondii mitochondrial proteome using MitoProt II. Proteins with high (>0.9; blue), medium (0.5 – 0.9; aqua) and low (<0.5; pink) prediction scores are shown.

Figure 3.. The localization of novel proteins from the T.gondii mitochondrial proteome.

(A–AA) Proteins with no previously determined localization in T. gondii were selected from the mitochondrial proteome, and the corresponding gene was tagged at the 3’-terminus of the open reading frame with a HA tag. Immunofluorescence assays depict HA-tagged proteins (green) co-labelled with the mitochondrial marker TgTom40 (red). The http://toxodb.org gene identification number is depicted for every gene that was tagged.

Figure 3—figure supplement 1.. Map of the pgCH plasmid vector, showing the SpeI, BglII and AvrII cut sites between which the 3’ flanks of target genes were ligated (green), the position of the 1x HA tag (yellow), and the positions of the chloramphenicol resistance marker (Chl^R) for T.gondii selection, the ampicillin resistance marker for E. coli selection (Amp^R), and the origin of replication (Ori; all magenta).

Note: vector is not drawn to scale.

Figure 4.. Orthology analyses of proteins from the T.gondii mitochondrial proteome reveal that many mitochondrial proteins are restricted to T. gondii and related organisms, and that most are important for parasite survival.

(A) Bar graph depicting the percentage of orthologs from the mitochondrial proteome of T. gondii (Tg) found in P. falciparum (Pf), B. bovis (Bb), C. parvum (Cp) and V. brassicaformis (Vb). Phenotype scores are indicated with shading, and reveal that most ortholog groups in each category are important or critical for tachyzoite growth. (B–C) Venn diagram depicting ortholog groupings from the mitochondrial proteome of T. gondii compared to (B) non-coccidian apicomplexans, chromerids and eukaryotes, or (C) non-coccidian apicomplexans, chromerids and animals. (D) Bar graph depicting distribution of phenotype scores in genes belonging to ortholog groups found only in T. gondii and other coccidians (Tg only), in T. gondii, non-coccidian apicomplexans and chromerids (Tg+ Api+ Chr), and in T. gondii, non-coccidian apicomplexans, chromerids and animals (Tg+ Api+ Chr+ Api). In (A) and (D), genes with phenotype scores of >-2 were considered dispensable, −2 to −4 were considered important, and <-4 were considered critical.

Figure 5.. TgApiCox25 is important for parasite growth and mitochondrial O₂ consumption.

(A) Western blot of proteins extracted from rTgApiCox25-HA parasites grown in the absence of ATc, or in ATc for 1–3 days, and detected using anti-HA antibodies (top) and anti-TgTom40 (as a loading control; bottom). (B) Plaque assays measuring growth of WT, rTgApiCox25 and complemented cTgApiCox25-HA/rTgApiCox25 parasites cultured in the absence (top) or presence (bottom) of ATc. Assays are from a single experiment and are representative of 3 independent experiments. (C) Quantification of plaque size from WT, rTgApiCox25 and complemented cTgApiCox25-HA/rTgApiCox25 parasites grown in the absence or presence of ATc for 9 days. Box and whisker plots depict the median plaque size (centre line), the 25^th and 75^th percentiles (box) and the 5^th and 95^th percentiles (lines). Data are from 30 plaques per flask from a single experiment, except in the case of the rTgApiCox25 strain, where only 18 plaques were discernible. (D) Basal mitochondrial oxygen consumption rates (mOCR) in WT parasites grown in the absence of ATc or in the presence of ATc for 3 days (orange), and rTgApiCox25 parasites grown in the absence of ATc, or in the presence of ATc for 1 – 3 days (blue). A linear mixed-effects model was fitted to the data, and the values depict the mean ± s.e.m. from three independent experiments. A one-way ANOVA followed by Tukey’s multiple pairwise comparison test was performed. Relevant p values are shown. (E) Basal mOCR plotted against basal extracellular acidification rate (ECAR) of WT cells grown in the absence of ATc, the presence of cycloheximide (CHX) for 1 day, or the presence of ATc for 3 days, and rTgApiCox25 parasites grown in the absence of ATc or presence of ATc for 1 – 3 days (mean ± s.e.m. of the linear mixed-effects model described above; n = 3).

Figure 5—figure supplement 1.. Generating an ATc-regulated promoter replacement strain of TgApiCox25.

(A) Diagram depicting the promoter replacement strategy to generated ATc-regulated TgApiCox25. A single guide RNA (sgRNA) was designed to target the T. gondii genome near the start codon of TgApiCox25, and mediate a double stranded break at the target site. A plasmid containing the sgRNA and GFP-tagged Cas9 endonuclease was co-transfected into T. gondii parasites with a PCR product encoding the ATc regulated ‘t7s4’ promoter, which contains 7 copies of the Tet operon and a Sag4 minimal promoter, flanked by 50 bp of sequence homologous to the regions immediately up- and down-stream of the TgApiCox25 start codon. The PCR product also contain a ‘spacer’ region that separates the regulatable promoter from the native promoter of TgApiCox25 gene to enable sufficient regulation. The parasite’s homologous repair pathway will mediate integration of the PCR product into the TgApiCox25 locus. The ‘TgApiCox25 fwd’, ‘TgApiCox25 rvs’ and ‘t7s4 fwd’ primers were used in screening parasite clones for successful integration of the regulatable promoter at the target site. (B–C) PCR screening analysis using genomic DNA extracted from parasite clones to identify clones that had successfully integrated the promoter. (B) Screening using the TgApiCox25 fwd and rvs primers. This will amplify a product of 1,064 bp if the locus is unmodified, and a product of 2,991 bp if the ATc-regulatable promoter has integrated successfully. (C) Screening using the TgApiCox25 rvs and t7s4 fwd primers. This will amplify a product of 928 bp if the ATc-regulatable promoter has integrated successfully. The analyses in B and C revealed that clone three had successfully integrated the ATc-regulatable promoter.

Figure 5—figure supplement 2.. Knockdown of TgApiCox25 leads to defects in maximal mOCR.

Maximal mOCR, comprising of the sum of the basal mOCR (colored) and the spare capacity (white), of TATi/∆ku80 (WT) parasites grown in the absence of ATc or in the presence of ATc for 3 days (orange), and rTgApiCox25 cells grown in the absence of ATc, or in the presence of ATc for 1–3 days (blue). A linear mixed-effects model was fitted to the data, which are depicted as the mean ±s.e.m. from three independent experiments. A one-way ANOVA followed by Tukey’s multiple pairwise comparison test was performed on the maximal mOCR values. Relevant p values are shown.

Figure 5—figure supplement 3.. Defects in mOCR upon TgApiCox25 knockdown are not the result of general defects in mitochondrial morphology or parasite viability.

(A) Immunofluorescence assays assessing mitochondrial morphology in rTgApiCox25 parasites grown in the absence of ATc (top) or in the presence of ATc for 3 days (bottom). Mitochondria were labelled using antibodies against TgTom40 (red). Images are representative of 100 four-cell vacuoles examined in two independent experiments. The scale bar is 2 µm. (B) Plaque assays of rTgApiCox25 parasites grown for 9 days in the absence (left) or presence (right) of ATc. rTgApiCox25 parasites were not preincubated in ATc (no ATc preinc; top) or pre-incubated in ATc for 3 days (3d + ATc preinc; bottom) before commencing the experiment. Plaque assays are from a single experiment, representative of 3 independent experiments.

Figure 5—figure supplement 4.. Map of the pUgCTH₃ plasmid vector, showing the BglII and AvrII cut sites between which the TgApiCox25 open reading frame was ligated (green), the position of the 3x HA tag (yellow) and α-tubulin 5’ region (blue), and the positions of the chloramphenicol resistance marker (Chl^R) for T.gondii selection, the UPRT flank (linearized at the indicated MfeI site before transfection), the ampicillin resistance marker for E. coli selection (Amp^R), and the origin of replication (Ori; all magenta).

Note: vector is not drawn to scale.

Figure 6.. TgApiCox25 is part of a 600 kDa protein complex and co-purifies with canonical components of the cytochrome c oxidase complex.

(A) Western blot of proteins extracted from TgApiCox25-HA parasites, separated by blue native-PAGE, and detected with anti-HA antibodies. (B) Western blot of proteins extracted from TgApiCox25-HA parasites, separated by SDS-PAGE, and detected with anti-HA antibodies. (C) Volcano plot showing the log₂ fold change vs –log₁₀ p values of proteins purified from TgApiCox25-HA vs TgTom40-HA parasites using anti-HA immunoprecipitations and detected by mass spectrometry. Only proteins detected in each of the three independent experiments for both parasite lines are depicted. Proteins enriched in the TgApiCox25-HA samples (p<0.05; log₂ fold change >5) have been coded according to whether they are orthologous to canonical cytochrome c oxidase subunits (green triangles), or restricted to the apicomplexan lineage (blue circles; ApiCox subunits). TgApiCox25 is also depicted (red diamond).

Figure 6—figure supplement 1.. Immunopurification of the TgApiCox25 and TgTom40 protein complexes.

(A–B) Western blots of proteins extracted from parasites expressing TgApiCox25-HA (A) or TgTom40-HA (B). Extracts include samples before immunoprecipitation (Total), samples that did not bind to the anti-HA beads (Unbound), and samples that bound to the anti-HA beads (Bound). Samples were probed with anti-HA (top) and anti-TgTom40 (bottom) antibodies. Immunoprecipitations are representative of three independent experiments. Bound fractions from each experiment were subjected to mass spectrometry-based protein identification.

Figure 7.. TgApiCox25 is a component of T.gondii cytochrome c oxidase and important for complex integrity.

(A) Anti-FLAG western blot of proteins from the TgCox2a-FLAG/TgApiCox25-HA strain separated by blue native-PAGE. (B) Western blots of proteins extracted from the TgCox2a-FLAG/TgApiCox25 HA strain and subjected to immunoprecipitation using anti-HA (anti-HA IP) or anti-FLAG (anti-FLAG IP) antibody-coupled beads. Extracts include samples before immunoprecipitation (Total), samples that did not bind to the anti-HA or anti-FLAG beads (Unbound), and samples that bound to the anti-HA or anti-FLAG beads (Bound). Samples were probed with anti-HA to detect TgApiCox25-HA, anti-FLAG to detect TgCox2a-FLAG, anti-AtpB to detect the β-subunit of T. gondii ATP synthase, and anti-TgTom40. (C) Anti-HA (left) and anti-FLAG (right) western blots of proteins from the TgApiCox25-FLAG/TgApiCox30-HA strain separated by blue native-PAGE. (D) Western blots of proteins extracted from the TgApiCox25-FLAG/TgApiCox30-HA strain and subjected to immunoprecipitation using anti-HA (anti-HA IP) or anti-FLAG (anti-FLAG IP) antibody-coupled beads. Extracts include samples before immunoprecipitation (Total), samples that did not bind to the anti-HA or anti-FLAG beads (Unbound), and samples that bound to the anti-HA or anti-FLAG beads (Bound). Samples were probed with anti-HA to detect TgApiCox30-HA, anti-FLAG to detect TgApiCox25-FLAG, anti-AtpB, and anti-TgTom40. (E) Western blot of proteins extracted from rTgApiCox25-HA/TgCox2a-FLAG parasites grown in the absence of ATc, or in ATc for 1 – 3 days, separated by SDS-PAGE and detected using anti-HA (top), anti-FLAG (middle) and anti-TgTom40 (as a loading control; bottom). (F) Western blot of proteins extracted from TgCox2a-FLAG/rTgApiCox25-HA parasites grown in the absence of ATc, or in ATc for 1 – 3 days, separated by blue native-PAGE, and detected using anti-FLAG antibodies.

Figure 7—figure supplement 1.. Generating FLAG tagged TgCox2a and TgApiCox25 strains.

(A) Diagram depicting the 3’ replacement strategy to generate FLAG-tagged TgCox2a. An sgRNA was designed to target the T. gondii genome near the stop codon of TgCox2a, and mediate a double stranded break at the target site. A plasmid containing the sgRNA and GFP-tagged Cas9 endonuclease was co-transfected into T. gondii parasites with a PCR product encoding a FLAG epitope tag flanked by 50 bp of sequence homologous to the regions immediately up- and down-stream of the TgCox2a stop codon. The parasite’s homologous repair pathway will mediate integration of the PCR product into the TgCox2a locus. Forward and reverse primers were used to screen parasite clones for successful integration of the FLAG tag at the target site, yielding a 260 bp product in the native locus and a 361 bp product in the modified locus. (B) PCR screening analysis using genomic DNA extracted from putative TgCox2a-FLAG/TgApiCox25-HA parasites (clones 1 – 10) and TgCox2a-FLAG/rTgApiCox25-HA parasites (clones 11 – 12). Clones 1 – 5 and 7 – 12 yielded PCR products that indicated that these clones had been successfully modified. (C) Diagram depicting the 3’ replacement strategy to generate FLAG-tagged TgApiCox25. An sgRNA was designed to target the T. gondii genome near the stop codon of TgApiCox25, and mediate a double stranded break at the target site. A plasmid containing the sgRNA and GFP-tagged Cas9 endonuclease was co-transfected into T. gondii parasites with a PCR product encoding a FLAG epitope tag flanked by 50 bp of sequence homologous to the regions immediately up- and down-stream of the TgApiCox25 stop codon. The parasite’s homologous repair pathway will mediate integration of the PCR product into the TgApiCox25 locus. Forward and reverse primers were used to screen parasite clones for successful integration of the FLAG tag at the target site, yielding a 385 bp product in the native locus and a 492 bp product in the modified locus. (B) PCR screening analysis using genomic DNA extracted from putative TgApiCox25-FLAG/TgApiCox30-HA parasites. Clones 1, 5 – 7 yielded PCR products that indicated that these clones had been successfully modified.