Divergent features of the coenzyme Q:cytochrome c oxidoreductase complex in Toxoplasma gondii parasites

Abstract

The mitochondrion is critical for the survival of apicomplexan parasites. Several major anti-parasitic drugs, such as atovaquone and endochin-like quinolones, act through inhibition of the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase complex (Complex III). Despite being an important drug target, the protein composition of Complex III of apicomplexan parasites has not been elucidated. Here, we undertake a mass spectrometry-based proteomic analysis of Complex III in the apicomplexan Toxoplasma gondii. Along with canonical subunits that are conserved across eukaryotic evolution, we identify several novel or highly divergent Complex III components that are conserved within the apicomplexan lineage. We demonstrate that one such subunit, which we term TgQCR11, is critical for parasite proliferation, mitochondrial oxygen consumption and Complex III activity, and establish that loss of this protein leads to defects in Complex III integrity. We conclude that the protein composition of Complex III in apicomplexans differs from that of the mammalian hosts that these parasites infect.

Fig 1. TgMPPα is part of a ~675 kDa protein complex and co-purifies with known components of Complex III.

(A) Western blot of proteins extracted from TgMPPα-TEV-HA parasites in 1% (w/v) digitonin, 1% (v/v) TX100 or 1% (w/v) DDM-containing lysis buffer, separated by BN-PAGE, and detected with anti-HA antibodies. (B) Western blot of proteins extracted from TgMPPα-TEV-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 TgMPPα-TEV-HA vs TgCox2a-TEV-HA parasites using anti-HA immunoprecipitations and detected by mass spectrometry. To enable statistical comparisons, only proteins detected in all three independent experiments for both parasite lines are depicted. Proteins enriched in the TgMPPα-TEV-HA samples (p < 0.05; log₂ fold change > 5) are labelled and shown in orange circles, with TgMPPα shown in red. Similarly, proteins previously identified as T. gondii Complex IV subunits [17] are shown in blue squares, with TgCox2a shown in red. (D) Table summarising the characteristics of proteins identified in proteomic analysis of the TgMPPα-TEV-HA complex (modelled after [18]). Proteins with homology to Complex III proteins are shown in orange circles, novel or divergent Complex III proteins are depicted by green circles, and proteins identified in the initial proteomic analysis but excluded in subsequent analyses are depicted in gray circles. Protein IDs were obtained from ToxoDB and proposed annotations are listed. The phenotype score (PS) of each gene predicts its importance for parasite proliferation, with scores < -2 typically found in genes that are important for proliferation [26]. Detection in the mitochondrial proteome (Mito Proteome, [17]) and its predicted cellular localisation (“LOPIT” [15]) are indicated (y = yes, n = no, N/A = not available, MM = mitochondrial membranes, O = outlier, N = nucleus, PM = plasma membrane). Homology indicates the tBLASTn expected value (E-value) between each T. gondii protein sequence and its closest match in Plasmodium falciparum (Pf), Vitrella brassicaformis (Vb), Chromera velia (Cv) or Cryptosporidium parvum (Cp) using EuPathDB searches. Black circles indicate a close match could not be identified. *, homology detected using HHPRED; #, homology detected using iterative JackHMMER searches.

Fig 2. Candidate Complex III subunits localize to the mitochondrion of T. gondii.

(Left) Western blot and (Right) immunofluorescence assay analysis of (A) TgMPPα-HA/TgQCR11-FLAG, (B) TgMPPα-HA/TgQCR8-FLAG, (C) TgMPPα-HA/TgQCR9-FLAG, or (D) TgMPPα-HA/TgQCR12-FLAG parasites. Western blots were detected with anti-FLAG antibodies, and immunofluorescence assays were detected with anti-FLAG (green) and the mitochondrial marker anti-TgTom40 (red) antibodies. Scale bars represent 2 μm.

Fig 3. Candidate Complex III subunits are part of a ~675 kDa protein complex and interact with TgMPPα.

(A) Western blot of proteins extracted from TgMPPα-TEV-HA (left) or TgMPPα-HA/TgQCR11-FLAG, TgMPPα-HA/TgQCR8-FLAG, TgMPPα-HA/TgQCR9-FLAG and TgMPPα-HA/TgQCR12-FLAG parasites in 1% (w/v) DDM, separated by BN-PAGE, and detected with anti-HA or anti-FLAG antibodies. Images were obtained from a single membrane with lanes cut as indicated and probed with different concentration of antibodies. (B-E) Western blots of proteins extracted from (B) TgMPPα-HA/TgQCR11-FLAG, (C) TgMPPα-HA/TgQCR8-FLAG, (D) TgMPPα-HA/TgQCR9-FLAG or (E) TgMPPα-HA/TgQCR12-FLAG parasites, 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 separated by SDS-PAGE, and probed with anti-HA antibodies to detect TgMPPα-HA, anti-FLAG to detect TgQCRs, and anti-TgTom40 as a control to detect an unrelated mitochondrial protein.

Fig 4. The apicomplexan-specific Complex III subunit TgQCR11 is important for parasite proliferation and mitochondrial oxygen consumption.

(A) Western blot of proteins extracted from rTgQCR11-FLAG/TgMPPα-HA parasites grown in the absence of ATc, or in the presence of ATc for 1–3 days, separated by SDS-PAGE, and detected using anti-FLAG and anti-TgTom40 antibodies (loading control). (B) Plaque assays measuring growth of WT, rTgQCR11-FLAG/TgMPPα-HA and complemented cTgQCR11-Ty1/rTgQCR11-FLAG/TgMPPα-HA parasites cultured in the absence (top) or presence (bottom) of ATc for 8 days. Assays are from a single experiment and are representative of 3 independent experiments. (C) Basal mitochondrial oxygen consumption rates (mOCR) of WT parasites grown in the absence of ATc or in the presence of ATc for 3 days (blue), and rTgQCR11-FLAG/TgMPPα-HA parasites grown in the absence of ATc or in the presence of ATc for 1–3 days (orange). A linear mixed-effects model was fitted to the data and values depict the least squares mean ± 95% CI of three independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed, with relevant p values shown. (D) Plaque assays of rTgQCR11-FLAG parasites grown in the absence of ATc (no ATc preinc; top) for 7 days, pre-incubated in ATc for 3 days before washing out and growing for a further 7 days in the absence of ATc (3d + ATc preinc; middle), or grown in the presence of ATc for all 7 days (7d + ATc; bottom). Quantifications of plaque number as a percent of the no ATc control are depicted to the right of the plaque assays, with bars representing the mean ± standard deviation of 3 independent experiments. (E) Plaque assays of WT parasites grown in the absence of atovaquone (no ATV preinc; top), pre-incubated in ATV for 2 days before washing out and growing for 7 days in the absence of ATV (2 days + ATV preinc; middle), or grown in the presence of ATV for all 7 days (7d + ATV; bottom). Quantifications of plaque number as a percent of the no ATV control are depicted to the right of the plaque assays, with bars representing the mean ± standard deviation of 3 independent experiments. (F) Basal mOCR versus basal extracellular acidification rate (ECAR) of WT parasites grown in the absence of ATc or in the presence of ATc for 3 days (blue), and rTgQCR11-FLAG/TgMPPα-HA parasites grown in the absence of ATc or in the presence of ATc for 1–3 days (orange). Data depict the mean mOCR and ECAR values ± 95% CI of the linear mixed-effects model (n = 3).

Fig 5. Loss of TgQCR11 leads to a specific defect in Complex III function.

(A) Schematic diagram of the assay measuring OCR in digitonin-permeabilized parasites, with inset (right) depicting a mock oxygen consumption rate (OCR) versus time graph to illustrate the typical response of WT parasites. Parasites are starved for 1 hour in base media to deplete endogenous energy sources, then permeabilized with 0.002% (w/v) digitonin before being subjected to the following injections of substrates or inhibitors: Port A, malate and glutamate (Mal/Glu) or glycerol 3-phopshate (G3P); Port B, antimycin A and atovaquone (AntA/ATV); Port C, TMPD and ascorbate (TMPD/Asc); Port D, sodium azide (NaN₃). The mitochondrial OCR (mOCR) elicited by a substrate (red line, mOCR^substrate) and the mOCR elicited by TMPD/Asc (blue line, mOCR^TMPD) are then calculated from these data. CytC, cytochrome c; CoQ, coenzyme Q; III, Complex III; IV, Complex IV; e^-, electrons. (B) Representative traces depicting OCR over time when supplying Mal/Glu (10 mM) as an energy source. WT (blue), rTgQCR11-FLAG/TgMPPα-HA (orange) and rTgApiCox25-HA (green) parasites were grown in the absence of ATc or in the presence of ATc for 1–3 days. Data represent the mean ± SD of three technical replicates, and are representative of three independent experiments. (C) Mal/Glu elicited mOCR (mOCR^Mal/Glu) of WT (blue), rTgQCR11-FLAG/TgMPPα-HA (orange) and rTgApiCox25-HA (green) parasites that were grown in the absence of ATc or in the presence of ATc for 1–3 days. A linear mixed-effects model was fitted to the data and values depict the least squares mean ± 95% CI from three independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed, with relevant p values shown. (D) Fold stimulation of mOCR by TMPD relative to Mal/Glu in WT (blue), rTgQCR11-FLAG/TgMPPα-HA (orange) and rTgApiCox25-HA (green) parasites that had been grown in the absence of ATc or in the presence of ATc for 1–3 days (mean ± 95% CI of the linear mixed-effects model; n = 3). ANOVA followed by Tukey’s multiple pairwise comparisons test was performed, with relevant p values shown. (E) Representative traces depicting OCR over time when supplying the TCA cycle-independent substrate G3P (10 mM) as an energy source. WT (blue) and rTgQCR11-FLAG/TgMPPα-HA (orange) parasites were grown in the absence of ATc or in the presence of ATc for 1–3 days. Data represent the mean ± SD of three technical replicates, and are representative of three independent experiments. (F) G3P elicited mOCR (mOCR^G3P) of WT (blue) and rTgQCR11-FLAG/TgMPPα-HA (orange) parasites that were grown in the absence of ATc or in the presence of ATc for 1–3 days (mean ± 95% CI of the linear mixed-effects model; n = 3). ANOVA followed by Tukey’s multiple pairwise comparisons test was performed, with relevant p values shown.

Fig 6. TgQCR11 is important for Complex III integrity.

(A-D) Western blots of proteins extracted from (A) rTgQCR11-FLAG/TgMPPα-HA, (B) rTgQCR11-FLAG/TgQCR12-TEV-HA, (C) rTgQCR11-FLAG/TgQCR8-TEV-HA and (D) rTgQCR11-FLAG/TgCytC1-HA parasites grown in the absence of ATc or in the presence of ATc for 1–3 days. Samples were prepared in 1% (w/v) DDM, separated by BN-PAGE, and detected with anti-HA antibodies. (E-H) Western blots of proteins extracted from (E) rTgQCR11-FLAG/TgMPPα-HA, (F) rTgQCR11-FLAG/TgQCR12-TEV-HA, (G) rTgQCR11-FLAG/TgQCR8-TEV-HA and (H) rTgQCR11-FLAG/TgCytC1-HA parasites that had been grown in the absence of ATc or in the presence of ATc for 1–3 days. Samples were separated by SDS-PAGE, and probed with anti-HA, anti-FLAG and anti-TgTom40 (loading control) antibodies. Western blots shown are representative of at least two independent experiments, with matched BN-PAGE and SDS-PAGE samples prepared from the same experiment. Quantifications of protein abundances are depicted in S13 Fig.