Molecular reagents and methods.

Genomic DNA and total RNA from tachyzoite stage T. gondii and plasmid DNA from recombinant E. coli (DH5α) were isolated using Qiagen kits (Germany); cDNA was prepared from total RNA using the reverse transcription kit from Thermo Fisher Scientific (United States of America). The manufacturer’s protocol was followed while using the various kits. PCR was done using a proof reading DNA polymerase obtained from Takara Bio (Japan), and all primers used in this study (S3 Table) were obtained from Integrated DNA Technologies (USA). Ligase and various restriction endonucleases were purchased from New England BioLabs (USA) or Promega (USA).

Plasmid constructs.

Various PCR products were cloned into the Topo 2.1 plasmid backbone (Thermo Fisher Scientific, USA) and sequenced before subcloning into other plasmids used for either modification of target genomic loci or ectopic expression of full-length cDNA for gene of interest in T. gondii. The plasmid construct used for tagging the 3′ end of F-type ATP synthase subunits β (Tgatpβ) and oscp (Tgatposcp) genes with YFP-HA tag had the following features: 2,606 bp (BglII and AvrII) and 1,831 bp (BamHI and AvrII) PCR fragments amplified from the 3′ end of Tgatpβ and Tgatposcp loci, respectively, excluding the stop codon, were cloned into the Topo 2.1 plasmid, upstream of a YFP-HA tag coding sequence, followed by the 3′ UTR of the T. gondii dihydrofolate reductase–thymidilate synthase (Tgdhfr-ts) gene [67]. A modified pBluescript plasmid backbone was used for ectopic expression of full-length cDNA of selected T. gondii genes encoding the novel F-type ATP synthase subunits identified from this work. The cDNAs were cloned as BamHI and XbaI/NheI fragments in frame to a HA tag for constitutive expression using the T. gondii β-tubulin gene promoter and Tgdhfr-ts gene 3′ UTR. For isolating clonal lines of transgenic parasites, the respective plasmid constructs include either the DHFR cassette (for endogenous tagging; expresses a mutant version of the Tgdhfr cDNA that confers pyrimethamine resistance [68]) or the CAT cassette (for ectopic expression; expresses the chloramphenicol acetyl transferase gene, which confers resistance to chloramphenicol [69]).

T. gondii culture and genetic manipulation.

Tachyzoite stage of T. gondii parasites (Type-I RH [RH] and Type-I RH ΔKu80 [RHΔKu80] [70]) strains were propagated following published protocols [71]. Briefly, human foreskin fibroblasts (HFFs), which were used as host cells for parasite infection, were grown in Dulbecco’s Modified Eagle Medium (DMEM High glucose) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM GlutaMAX, 25 mM HEPES, and 50 μg/ml Gentamicin. All cell culture grade reagents were procured from Thermo Fisher Scientific, USA. The HFF cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. T. gondii tachyzoites were propagated by inoculating approximately 10⁵ freshly isolated tachyzoites into T25 flasks containing 2-week-old confluent HFF cell monolayer. The culture medium used for parasite growth is similar to that used for host cell growth except that it lacks serum. Parasites are harvested 48 hours after infection by scrapping the host cell monolayer, physically disrupting the HFF cell suspension by passing it repeatedly through a 22-gauge needle, and filtering it through a 3 μM nucleopore membrane (Whatman, GE Healthcare, USA) to obtain a suspension of parasites devoid of HFF cell debris. The number of parasites present in 1 ml of suspension is estimated from cell counts obtained using a hemocytometer.

For generating transgenic parasites expressing the TgATPβ and TgATPOSCP proteins from the endogenous loci, the RHΔKu80 parasites were transfected [70] with the respective tagging plasmids. Transfection was achieved by electroporating 10⁷ freshly harvested tachyzoite-stage parasites resuspended in 400 μl parasite culture medium containing 50 μg of linearized sterile plasmid DNA, using a BioRad (USA) Gene Pulser system using 10 μF capacitance, ∞ ohms resistance, and 1.5 kV voltage settings. Transfected parasites were immediately inoculated into a T25 flask containing HFF monolayer and allowed to invade and replicate for 12–15 hours before beginning drug selection with 1 μM pyrimethamine. For ectopic expression of cDNA with HA tags for selected genes, RH parasites were transfected with the respective plasmid constructs, and stable transformants were selected using 20 μM chloramphenicol. Clonal lines of stable transgenic parasites were isolated using the limiting dilution technique [71].

Measurement of intracellular ATP.

For this assay, freshly isolated extracellular tachyzoite-stage parasites were incubated in culture media that either contained 5.5 mM glucose and 4 mM glutamine or 0 mM glucose and 4 mM glutamine, in order to evaluate the ability of the parasites to maintain ATP homeostasis. The antiparasitic drug atovaquone [43], an inhibitor of mtETC, was used to specifically inhibit mitochondrial ATP synthesis. After 2 hours of incubation, total cellular ATP was measured using the ViaLight Plus Cell Proliferation and Cytotoxicity Bioassay Kit (Lonza, Switzerland) as per the manufacturer’s protocol. The luminescence readout was quantified using the Varioskan Flash plate reader (Thermo Fisher Scientific, USA).

Microscopy and western blotting.

For visualizing the expression and subcellular localization of selected ASAPs fused to a reporter tag, replicating intracellular tachyzoite-stage parasites stably expressing these proteins were imaged by microscopy. Before imaging, the parasite-infected HFF monolayers cultured on glass coverslips were washed with 1× phosphate-buffered saline (PBS) and fixed using 4% paraformaldehyde for 20 minutes before again washing and mounting on glass slides using the fluoroshield reagent (Sigma, USA). To counterstain the parasite mitochondrion, the cells were treated with 250 nM Mitotracker Red (Thermo Fisher Scientific, USA) for 20 minutes before fixing. The slides were imaged using the 63× oil immersion objective fitted to the Axio Observer inverted fluorescent microscope (Carl Zeiss, Germany), and images were processed using the Zen software (Carl Zeiss, Germany). Intrinsic fluorescence from YFP and Mitotracker Red were visualized using the excitation/emission filter combination of 493/520 and 578/599, respectively. HA-tagged proteins were visualized by immunofluorescence staining, using rabbit α-HA primary antibodies (1:1,000) followed by Alexa 488 conjugated goat anti rabbit secondary antibody (1:1,000), both purchased from Thermo Fisher Scientific, USA. After fixing, the cells were permeabilized for 5–10 minutes using 0.25% Triton X-100 in 1× PBS. They were then treated with 2% fetal bovine serum in 1× PBS for 30 minutes, followed by primary antibody for 1 hour, 3× wash with 1× PBS containing 0.25% Triton X-100, and secondary antibody for 1 hour. Finally, the cells were washed 3× with 1× PBS containing 0.25% Triton X-100, followed by a water wash, before mounting the cover slip on glass slides for imagining.

For western blotting, purified mitochondria were solubilized in 1× Laemmli buffer (120 mM Tris-HCL pH 6.8, 2% SDS, 10% glycerol, and 0.01% w/v bromophenol blue) and resolved by SDS-PAGE, followed by electro-transfer to PVDF membrane (using Tris-glycine buffer pH 8 containing 20% methanol) at 65 mA for approximately 1 hour and 20 minutes. The blots were kept in blocking buffer (5% skimmed milk in 1× PBS) overnight at 4 °C and then treated with primary antibody (1:5,000; rabbit α-HA monoclonal antibody, Thermo Fisher Scientific, USA) for 1 hour at ambient temperature. This was followed by 3× wash with 1× PBS containing 0.1% Tween 20, before incubating with horseradish peroxidase–coupled secondary antibody (1:5,000; donkey anti-rabbit antibody, Nif 824 GE Healthcare, USA) for 1 hour at ambient temperature, and 3× wash with 1× PBS containing 0.1% Tween 20. The blot was then developed using either the 3,3’-diaminobenzidine substrate (Sigma, USA) or the chemiluminescent ECL western blotting kit (GE Healthcare, USA).

Preparation of mitochondria from T. gondii.

Freshly harvested tachyzoite-stage transgenic parasites (approximately 10⁹) expressing either TgATPβ-YFP-HA or TgATPOSCP-YFP-HA were washed with PBS and resuspended in 1–2 ml of hypotonic lysis buffer (15 mM phosphate buffer pH 7.5 and 2 mM Glucose). The cells were lysed by sonication in an iced-water bath for 30 minutes, and samples were centrifuged at 2,000 g for 2 minutes at 4 °C to remove unbroken cells and large cell debris. The supernatant was recovered into a new tube and centrifuged at 21,000 g for 15 minutes at 4 °C to pellet the mitochondria, which was then resuspended in 500 μl of mitochondria storage buffer (320 mM sucrose, 1 mM EDTA, 10 mM Tris pH 7.4). Total protein in the mitochondria suspension was estimated by Bradford method [72], and the lysate was stored in −80 °C until further use. When required, the mitochondria were recovered from storage buffer by centrifuging at 21,000 g for 15 minutes at 4 °C and lysed using mitochondria solubilization buffer A (MSB-A; 50 mM NaCl, 50 mM Imidazole, 2 mM 6-Aminohexoanic acid, 1 mM EDTA, pH 7.0) containing β-dodecyl maltoside (DDM) detergent in total protein to detergent ratio of 1:5. Solubilization was allowed to continue overnight with rocking at 4 °C, and the solubilized fraction was separated by centrifuging at 100,000 g for 20 minutes at 4 °C.

BNP separation, in-gel ATPase activity assay, and western blotting.

In order to identify the monomeric and dimeric forms of the T. gondii F-type ATP synthase, the solubilized mitochondrial lysate was subject to one-dimensional BNP analysis [73,74]. Samples were prepared by adding glycerol to a final concentration of 5%, and Coomassie Blue G 250 5% (w/v) dye was added such that DDM detergent to dye ratio was 8:1. Samples were separated on a 3%–12% native PAGE (Thermo Fisher Scientific, USA) at 150 V setting for 30 minutes in cathode buffer A, which was then switched to cathode buffer B, and separation was continued till the blue dye exited the gel. The gel was visualized under white light to identify the various dye stained bands. Imidazole (25 mM, pH 7.0) was used as the anode buffer, while the composition of cathode buffers A and B were 50 mM Tricine, 7.5 mM imidazole pH 7.0, 0.02% Coomassie Blue G 250 dye and 50 mM Tricine, 7.5 mM imidazole pH 7.0, 0.002% Coomassie Blue G 250 dye, respectively.

For in-gel ATPase assay, after sample separation by BNP, the gel was incubated overnight in assay buffer (35 mM Tris-HCL pH 7.8, 270 mM glycine, 14 mM MgSO4, 0.2% Pb(NO₃)₂, and 8 mM ATP). The gel was then washed with water, and the ATPase activity of F-type ATP synthase was observed by the formation of a milky white precipitate, visible against a black background, at the region corresponding to the expected monomeric and dimeric protein bands. For western blotting after BNP separation, proteins were electro-transferred onto a PVDF membrane using Tris-glycine buffer pH 8.0 without methanol at 65 mAh for approximately 1 hour and 20 minutes. The membrane was then fixed in 8% acetic acid for 15 minutes, air dried at room temperature for 30 minutes, and washed with methanol several times to remove the Coomassie dye stain [26]. Further steps were similar to that described above for SDS PAGE western blotting.

IP of F-type ATP synthase complex.

The supernatant from solubilized mitochondria preparation from tachyzoite stage transgenic T. gondii expressing YFP-HA tagged TgATPOSCP subunit was used for immunoprecipitating the F-type ATP synthase using α-HA antibodies (Thermo Fisher Scientific, USA). The antibodies were first cross-linked to Protein A/G Plus agarose beads (Santa Cruz Biotechnology, USA) using the following protocol. Approximately 50 μl of Protein A/G Plus agarose beads were washed twice in 500 μl 1× PBS (pH 7.4) at 4 °C and mixed with 1 ml of 1× PBS containing 5–7 μg of rabbit α-HA antibody and incubate overnight at 4 °C with gentle mixing on rocker. The beads were then separated by centrifugation (1,000 g for 2 minute at 4 °C), equilibrated with 0.2 M Triethanolamine (pH 8.2) for 2 minutes, and then washed twice with the same buffer. The bound antibodies were then cross-linked to the beads using 20 mM DMP (Sigma, USA) in 1 ml of 0.2 M Triethanolamine at room temperature for 45 minutes, followed by washing with 1 ml of 50 mM Tris pH 7.5 twice for 15 minutes to quench the cross-linking reaction. Unbound antibodies are removed using 3 quick washes with 0.2 M glycine buffer pH 2.3. The antibody conjugated beads were then washed twice with 1× PBS and equilibrate with MSB containing the detergent DDM at critical micelle concentration. The supernatant from solubilized mitochondria was added to the antibody coupled beads and incubated overnight at 4 °C with gentle mixing on rocker. The beads were then washed 3 times with 1× PBS containing DDM at critical micelle concentration. Proteins captured by the antibodies were eluted in 100 μl of 0.2 M glycine buffer (pH 2.3) in 3 rounds, and the pooled eluate was neutralized with 1 M Tris (pH 8.0) and concentrated using a 3 KDa cut-off concentrator (Amicon Millipore, Germany). The samples were equilibrated with 0.1% RapiGest (Waters, USA) in 50 mM ammonium bicarbonate buffer in preparation for LC-MS analysis.

Purification of native F-type ATP synthase by chromatography.

We followed a protocol that was previously reported for the purification of the F-type ATP synthase from the Polytomella sp. [75]. About 13 mg (wet weight) of mitochondrial preparation from transgenic T. gondii expressing YFP-HA tagged TgATPβ subunit was solubilized overnight in 3 ml of mitochondrial solubilization buffer B (MSB-B; 50 mM Tris HCl [pH 8.0], 1 mM MgCl₂, and DDM at protein to detergent ration 1:5). The supernatant was loaded onto a HiTrap DEAE sepharose column (GE Healthcare Life sciences, USA) having a bed volume of 1 ml and equilibrated with buffer A (50 mM Tris HCl [pH 8.0] MgCl₂ 1mM, 0.05% [w/v] DDM). After completion of loading, the column was first washed with 10 column volumes of buffer B₂₀ (50 mM Tris HCl [pH 8.0], 1 mM MgCl₂, 20 mM NaCl, and 0.05% [w/v] DDM) and eluted with a 10× column volume linear gradient using buffer B₂₀ and buffer B₅₀₀ (50 mM Tris HCl [pH 8.0], 1 mM MgCl₂, 500 mM NaCl, and 0.05% [w/v] DDM) at a flow rate of 0.5 ml per minute. Fractions (500 μl) were collected and checked by western blotting using α-HA antibodies. Peak positive fractions were identified, pooled, and concentrated to a final volume of 200 μl using the Vivaspin concentrator (100 kDa cut-off; GE Healthcare Life sciences, USA) and separated by size exclusion chromatography using the Superose 6 Increase 3.2/300 column (GE Healthcare Life sciences, USA) having a bed volume of 2.4 ml. A 50 μl sample was loaded on the column previously equilibrated with buffer B₂₀ and then eluted with 1.5 column volumes of the same buffer at a 50 μl flow rate. Fractions (100 μl) were collected, and positive fractions, identified by western blotting using α-HA antibodies, were pooled and concentrated using a 3 KDa cut-off concentrator (Amicon Millipore, Germany). Samples were then equilibrated with 0.1% RapiGest in 50 mM ammonium bicarbonate buffer in preparation for LC-MS analysis. All chromatography steps were carried out using the AKTApure system (GE Healthcare Life sciences, USA).

LC-MS proteomics.

In order to identify the subunit components of the F-type ATP synthase from T. gondii, we performed LC-MS analysis on the partially purified enzyme obtained from BNP, IP, and chromatographic purification. Sample preparation for LC-MS analysis was based on a previous report [76]. The bands corresponding to the dimer and monomer forms of the protein were excised out from BNP gel, cut to small pieces, destained with 50% acetonitrile in 50 mM ammonium bicarbonate, and dehydrated with 100% acetonitrile. The gel pieces were then treated with 10 mM DTT in 50 mM ammonium bicarbonate for 45–60 minutes at 56 °C to reduce the proteins, followed by alkylation in the dark with 55 mM Iodoacetamide in 50 mM ammonium bicarbonate at ambient temperature for 45 minutes. Then, trypsinization of protein was carried out overnight at 37 °C, and peptides were extracted using 50% acetonitrile in 2% formic acid. The extracts were dried using a speedvac, and the peptides were reconstituted in 50 μl of 50 mM ammonium bicarbonate, acidified with HCl, and desalted using the C₁₈ Zip tip columns (Millipore, Germany). The samples were then dried in a speedvac and stored at −80 °C until further use.

Samples obtained from IP and size exclusion chromatography were immediately equilibrated with 0.1% RapiGest as stated above. The mixtures were then heated to 80 °C and maintained at this temperature for 15 minutes. The denatured proteins were reduced by 100 mM DTT at 60 °C for 15 minutes, followed by alkylation with 200 mM iodoacetamide at ambient temperature and in the dark for 30 minutes. Samples were then treated overnight with trypsin (Sigma, USA) at 20:1 substrate to enzyme ratio at 37 °C. Trypsinization was stopped by addition of 2 μl of 1 N HCL and incubated at 37 °C for 20 minutes, after which they were vortexed and centrifuged. The peptide samples were desalted using the C₁₈ Zip tip (Millipore, Germany), dried, and stored at −80 °C until further use.

The peptide samples were analyzed using LC-MS^E workflow on the nano ACQUITY (UPLC) Synapt system (Waters Corporation, USA). The digested peptide sample (4 μl) was injected into a 5 μm Symmetry C₁₈ trapping column (180 μm × 20 mm) at a flow rate of 5 μl/minute, and peptides were eluted by using the following protocol: 3%–40% B (B composition: 100% acetonitrile with 0.1% formic acid) for 90 minutes, 40%–85% B from 90–105 minutes and 97% A (A composition: 0.1% formic acid in water), and 3% B from 105–120 minutes, on a bridge ethyl hybrid C₁₈ column (75 μm × 250 mm, 1.7 μm) at flow rate of 250 nl/minute. The system was coupled to the Synapt High Definition Mass Spectrometer (Waters Corporation, USA) with a nonoLock spray as the ion source. Standard glu-fibrinopeptide B (Sigma, USA) as the lockmass calibrate peptide was infused into ion source having 500 nl/minute flow rate and sampled every 30 seconds. Acquisition of LC-MS^E data was performed in positive V mode with mass range m/z 50–1,900, having a scan time of 0.75 second, a constant low energy of 4 V for MS mode, and 20–40 V of collision energy during high-energy MS^E mode scan. A capillary voltage of 3.5 kV and cone voltage 38 V were maintained during analysis. LC-MS^E data were processed using Protein Lynx Global Server (PLGS Version 2.5.3, Waters Corporation, USA) for identifying the proteins, with reference to the T. gondii proteome taken from ToxoDB.org release 36.

Gene coexpression analysis and phylogenetic studies.

Normalized signal intensity values from microarray hybridizations performed at 13 different time points in the tachyzoite stage [52] were obtained directly from EupathDB.org. Normalized microarray intensities from time series data for intraerythrocytic stage P. falciparum were retrieved from reference [53]. Pearson correlation coefficients were calculated for all gene pairs using R (R-project.org). P-values were calculated using the Mann-Whitney U test [77] for coexpression between ATP synthase genes versus coexpression between ATP synthase genes and non-ATP synthase genes and for coexpression between ATP synthase genes versus coexpression between non-ATP synthase genes. Since minute differences in very large samples may lead to very small p-values, the effect size was therefore tested using Cohen’s d test [78], for which values above 0.8 denote large effect size. Orthologs for the novel subunits of T. gondii ATP synthase were identified, as reported previously [42]. Sequences were aligned with mafft (v7.222) [79] and Neighbor-Joining trees were constructed using Clustalw (2.1) [80] using 1,000 bootstrap replicates. The expected “true” phylogeny was adopted from a previous study [42]. The tree data have been deposited in TreeBASE (TB2:S22877).