Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi

Chemicals and reagents.Heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin were from Vitrocell (Campinas, Brazil). G418 was from KSE Scientific (Durham, NC). Puromycin, blasticidin S HCl, Platinum Taq DNA polymerase high fidelity, subcloning efficiency DH5α competent cells, and MitoTracker Deep Red FM were from Invitrogen (Eugene, OR). Antarctic phosphatase, BamHI, XbaI, XhoI, and HindIII restriction enzymes were from New England BioLabs (Ipswich, MA). T4 DNA ligase, GoTaq G2 Flexi DNA polymerase, Wizard Plus SV miniprep purification system, Wizard SV gel, and PCR clean-up system were from Promega (Madison, WI). Four-millimeter electroporation cuvettes, Zeta-Probe GT membrane, Precision Plus protein dual color standards, and nitrocellulose membranes were from Bio-Rad (Hercules, CA). North2South chemiluminescent hybridization and detection kit, North2South biotin random prime labeling kit, bicinchoninic acid (BCA) protein assay kit, SuperSignal West Pico chemiluminescent substrate, mouse anti-HA monoclonal antibody, and calcium green-5N were from Thermo Fisher Scientific (Waltham, MA). Mouse anti-c-Myc monoclonal antibody (9E10) was from Santa Cruz Biotechnology (Dallas, TX). Horseradish peroxidase-conjugated anti-mouse IgG antibody and Alexa Fluor 488-conjugated goat anti-mouse were from Life Technologies (Grand Island, NY). Fluoromount-G mounting medium was from Southern Biotech (Birmingham, AL). UK5099 was from Tocris Bioscience (Abingdon, United Kingdom). The pMOTag23M vector (50) was a gift from Thomas Seebeck (University of Bern, Bern, Switzerland). DNA oligonucleotides were purchased from Exxtend Biotecnologia Ltd. (Campinas, Brazil). Phenylmethylsulfonyl fluoride (PMSF), N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E64), protease inhibitor cocktail for use with mammalian cell and tissue extracts (catalog number P8340), mouse anti-tubulin monoclonal antibody, digitonin, ADP, oligomycin A, carboxyatractyloside (CAT), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, safranine O, malonate, sodium pyruvate, succinate, α-ketoglutarate, Benzonase nuclease, and all other reagents of analytical grade were from Sigma-Aldrich (St. Louis, MO).

Cell cultures.T. cruzi epimastigotes (Y strain) were grown at 28°C in liver infusion tryptose (LIT) medium (51) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U ml⁻¹), and streptomycin (100 μg ml⁻¹). LIT medium contained 2 g liter⁻¹ glucose. To prepare a low-glucose LIT medium, glucose was not added. The glucose in the supplemented low-glucose LIT medium (approximate glucose concentration of 0.07 g liter⁻¹) was from the FBS. Cell density was verified by counting parasites using Muse cell analyzer (Millipore, Burlington, MA). Parasites from the control cell lines transfected with scrambled sgRNA (scrambled) or pTREX-n-3×HA empty vector (EV), and overexpressing (OE) cell lines TcMPC1-OE and TcMPC2-OE were grown in the presence of 250 μg ml⁻¹ G418. TcMPC1-TAG and TcMPC2-TAG cell lines were maintained with 250 μg ml⁻¹ G418 and 5 μg ml⁻¹ puromycin. TcMPC1-KO and TcMPC2-KO cell lines were grown with 250 μg ml⁻¹ G418 and 10 μg ml⁻¹ blasticidin. Tissue culture-derived trypomastigotes were collected from the culture medium of infected Vero cells, using a modification of the method of Schmatz and Murray (1982) as previously described (52). Vero cells were grown in RPMI medium supplemented with 10% FBS and maintained at 37°C with 5% CO₂.

In silico analyses for selection of protospacers.For knockout and tagging of TcMPC genes, we used the genome editing method by CRISPR/Cas9 for T. cruzi developed before (26, 28, 53). Selection of protospacers for both strategies was performed using EuPaGDT (eukaryotic pathogen CRISPR guide RNA/DNA design tool; http://grna.ctegd.uga.edu) (54).

Endogenous C-terminal tagging of TcMPCs.CRISPR/Cas9-mediated endogenous C-terminal tagging of TcMPC proteins was performed as previously described (26, 27). To perform individual tagging of TcMPC1 and TcMPC2, we chose protospacer targeting regions close to the 3′ end of the putative TcMPC1 gene (294 bp, TriTrypDB gene ID, TcCLB.511577.144) and the putative TcMPC2 gene (414 bp, TriTrypDB gene ID, TcCLB.508265.44). sgRNAs were amplified by PCR using a forward oligonucleotide (primers 1 and 2, Table S1 in the supplemental material) and a common reverse primer (Rv_sgRNA, primer 3, Table S1) and further cloned into the Cas9/pTREX-n vector. To amplify DNA donor cassettes containing 3×c-Myc tag sequence and marker for puromycin resistance (Pac gene), a set of long primers (ultramers) (primers 5 to 8, Table S1) for each gene was designed. DNA donors were amplified by PCR using pMOTag23M vector as the DNA template. Wild-type T. cruzi epimastigotes (Y strain) were cotransfected with the gene-specific sgRNA/Cas9/pTREX-n plasmid and linear DNA donor. After 6 weeks of selection with G418 and puromycin, tagging of TcMPC1 and TcMPC2 was confirmed by PCR analysis using specific primer sets (primers 9 to 16, Table S1) and by Western blotting. A scrambled sgRNA cell line was used as control (53).

Knockout of TcMPCs.Molecular construction of sgRNA/Cas9/pTREX-n vector was performed as previously described (28). For knocking out TcMPC1 and TcMPC2 individually, we chose protospacer sequences closest to the 5′ end of the gene of interest (GOI), and sgRNAs were amplified by PCR using a forward oligonucleotide (primers 17 and 18, Table S1) and a common reverse primer (Rv_sgRNA, primer 3, Table S1). Ultramers (primers 19 to 22, Table S1) for each gene were designed to amplify DNA donors containing the blasticidin resistance gene (Bsd) using pGEM-Bsd vector as the DNA template. Wild-type T. cruzi epimastigotes (Y strain) were cotransfected with TcMPC-specific sgRNA/Cas9/pTREX-n vector and linear DNA donor. A scrambled sgRNA cell line was used as the control. After 6 weeks of selection with G418 and blasticidin, knockout of TcMPC1 and TcMPC2 was confirmed by PCR analysis using specific primer sets (primers 23 to 32, Table S1) and by Southern blotting.

Overexpression of TcMPC proteins.For overexpression of 3×HA-tagged TcMPC1 and TcMPC2 proteins, the primers 33 to 36 (Table S1) were designed (XbaI and XhoI restriction sites in the primers forward and reverse, respectively). The pTREX-n/3×HA vector, which provides resistance to G418, and PCR products were doubly digested with XbaI and XhoI. The inserts were independently cloned into the digested pTREX-n/3×HA vector using T4 DNA Ligase, generating pTREX-n/TcMPC1-3×HA and pTREX-n/TcMPC2-3×HA plasmids. The recombinant constructs were confirmed by PCR analysis with Fw_HX1-pTREX primer (primer 37, Table S1) and the gene-specific reverse primer, digestion with the restriction enzymes, and sequencing. Wild-type T. cruzi epimastigotes (Y strain) were transfected with recombinant constructs. Epimastigotes transfected with pTREX-n/3×HA empty vector were used as the control. After 6 weeks of selection with G418, overexpression of TcMPC1 or TcMPC2 was confirmed by Western blot analysis.

Cell transfections.Y strain wild-type T. cruzi epimastigotes were used for cotransfection of the sgRNA/Cas9/pTREX-n plasmid and linear DNA donor. Cell transfections were performed as reported previously (53) in triplicates. Negative controls were flasks containing cells without plasmid DNA that were subjected to the electroporation, and flasks containing cells without plasmid DNA that were not subjected to electroporation.

Transfectants TcMPC1-TAG and TcMPC2-TAG were cultured in LIT medium supplemented with 20% heat-inactivated FBS, penicillin, and streptomycin plus 250 μg ml⁻¹ G418 and 5 μg ml⁻¹ puromycin until stable cell lines were obtained. Transfectants TcMPC1-KO and TcMPC2-KO were cultured in the same medium plus 250 μg ml⁻¹ G418 and 10 μg ml⁻¹ blasticidin until stable cell lines were obtained.

For transfection with the overexpression vectors pTREX-n/TcMPC1-3×HA and pTREX-n/TcMPC2-3×HA, we followed the protocol described above using 25 μg of the plasmid. Transfectants TcMPC1-OE and TcMPC2-OE were cultured in LIT medium supplemented with 20% heat-inactivated FBS, penicillin, and streptomycin plus 250 μg ml⁻¹ G418.

Southern blot analyses.To corroborate TcMPC1 and TcMPC2 gene knockouts by Southern blotting, 25 μg of gDNA from scrambled sgRNA and TcMPC1-KO cell lines was digested with HindIII enzyme and 25 μg of gDNA from scrambled sgRNA and TcMPC2-KO cell lines was digested with BamHI enzyme. Products of restriction enzyme digestion were resolved on a 1% agarose gel that was further subjected to alkaline blotting using a nylon membrane (Zeta-Probe GT membrane) according to the manufacturer’s recommendations. The next steps were performed with a North2South chemiluminescent hybridization and detection kit. Probes for hybridization were amplified by PCR using the same primer sets used for the overexpression strategy (primers 33 to 36, Table S1) and pTREX-n/TcMPC1-3×HA or pTREX-n/TcMPC2-3×HA vector as the DNA template. DNA probes were biotinylated using North2South biotin random prime labeling kit.

Western blot analyses.To confirm the expression of the proteins TcMPC1-3×c-Myc, TcMPC2-3×c-Myc, TcMPC1-3×HA, and TcMPC2-3×HA, mid-log-phase epimastigotes from the respective cell lines TcMPC1-TAG, TcMPC2-TAG, TcMPC1-OE, and TcMPC2-OE were harvested by centrifugation at 1,000 × g for 7 min and washed twice with phosphate-buffered saline (PBS), pH 7.4, at room temperature (RT). The pellets were resuspended in 100 μl of RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], and 0.1% Triton X-100) plus 1 mM PMSF, 2.5 mM TPCK, 100 μM E64, and protease inhibitor cocktail (P8340, 1:250 dilution). Samples were incubated on ice for 1 h and then homogenized with a 1-ml syringe. Lysates were kept at −80°C until further use. Protein concentration in the lysates was determined using the BCA protein assay kit. Approximately 50 μg of each lysate was mixed with 6× Laemmli sample buffer (300 mM Tris-HCl, pH 6.8, 6% [wt/vol] β-mercaptoethanol, 50% [vol/vol] glycerol, 12% [wt/vol] SDS, 4% [wt/vol] bromophenol blue) and then subjected to electrophoresis using a 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Precision Plus protein dual color standard was applied on the gel. Electrophoresed proteins were transferred to a nitrocellulose membrane using a Bio-Rad transblot apparatus for 1 h at 100 V at 4°C. Following transfer, the membrane blots were blocked with 5% nonfat dry milk in PBS containing 0.1% (vol/vol) Tween 20 (PBS-T) at 4°C overnight. Blots were probed with mouse anti-c-Myc monoclonal antibody (1:100 dilution in PBS-T), mouse anti-HA monoclonal antibody (1:5,000 dilution in PBS-T), or mouse anti-tubulin monoclonal antibody (1:40,000 dilution in PBS-T) for 1 h at RT. After washing three times with PBS-T, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:10,000 dilution in PBS-T) for 1 h at RT. The membranes were washed three times with PBS-T, and Western blot images were processed and analyzed using SuperSignal West Pico chemiluminescent substrate and a UVItec Cambridge Alliance 2.7 imaging system (UVItec, Cambridge, United Kingdom) according to the manufacturer’s instructions.

For Western blot analysis to detect phosphorylation of PDH, 30 μg of each lysate, the rabbit polyclonal anti-PDH-E1α (phospho-Ser-293) primary antibody (1:1,000 dilution in PBS-T), and the goat HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000 dilution in PBS-T) were used.

Immunofluorescence analyses.For immunofluorescence assays of TcMPC1-TAG, TcMPC2-TAG, TcMPC1-OE, and TcMPC2-OE epimastigotes, cells were incubated with 100 nM MitoTracker Deep Red FM in culture medium for 30 min at 28°C. After staining with MitoTracker dye, cells were washed twice with PBS, pH 7.4 (PBS1), and fixed with 4% paraformaldehyde in PBS1 for 1 h at RT. Then, cells were allowed to adhere to poly-l-lysine-coated coverslips for 30 min. The coverslips were washed three times with PBS1 and incubated with 0.3% Triton X-100 in PBS1 for 3 min. Permeabilized cells were washed twice with PBS1 and blocked with PBS1 containing 5% goat serum, 50 mM NH₄Cl, 3% bovine serum albumin (BSA), and 1% fish gelatin at 4°C overnight. After blocking, cells were incubated with a primary antibody, mouse anti-c-Myc monoclonal antibody (1:10 dilution, 1% BSA in PBS, pH 8.0 [PBS2]) for TcMPC-TAG epimastigotes or mouse anti-HA monoclonal antibody (1:500 dilution in PBS2) for TcMPC-OE epimastigotes, for 1 h at RT. The coverslips were washed three times with 1% BSA in PBS2 and then incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody (1:1,000 dilution) diluted in 1% BSA in PBS2 for 1 h at RT in the dark. Cells were washed three times with 1% BSA in PBS2, washed once with PBS2, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 5 μg ml⁻¹) in Fluoromount-G mounting medium on the slides. Negative controls were performed as described above but in the absence of the primary antibody. Differential interference contrast and fluorescence optical images were captured with a 100× oil immersion lens objective (1.44 aperture) under nonsaturating conditions with a Leica TCS SP5 II confocal microscope, which uses photomultiplier tubes for the detection of emission. Digital images were acquired and processed with Leica LAS AF Lite software (Leica, Wetzlar, Germany).

Metacyclogenesis assay.The protocol described by Bourguignon et al. (55) was followed with minor modifications (20). Cells were harvested from TAU 3AAG medium at day 5 of cultivation. Three independent experiments were conducted.

Invasion and replication assays.Gamma-irradiated (2,000 radiation-absorbed doses) Vero cells (4.5 × 10⁵ cells) were plated onto sterile coverslips in a 12-well plate and incubated overnight at 35°C with 7% CO₂ in RPMI medium supplemented with 10% fresh FBS. Tissue culture-derived trypomastigote collections were incubated at 4°C overnight to allow amastigotes to settle from swimming trypomastigotes. Trypomastigotes from the supernatants of these collections were counted and used to infect the mammalian cells at a 10:1 ratio of parasites to host cells. At 4 h postinfection, coverslips were washed extensively with Hank’s balanced salt solution, followed by PBS, pH 7.4, to remove any extracellular parasites. This protocol has been optimized with a multiplicity of infection (MOI) of 10 to ensure that, at most, one trypomastigote (Y strain) invades each irradiated host cell. Coverslips were immediately fixed in 4% paraformaldehyde in PBS, pH 7.4, at 4°C for 30 min and then washed once with PBS and counterstained with DAPI (15 μg ml⁻¹) in Fluoromount-G mounting medium on glass slides, which stains host and parasite DNA. Coverslips were analyzed on an Olympus BX60 microscope to quantify the number of host cells that contained intracellular parasites and the number of intracellular parasites per cell in 40 randomly selected fields. Four hundred host cells were counted per sample in three independent experiments. To quantify amastigote replication, the following modifications were used: Vero cells were infected at a 10:1 ratio of parasites to host cells, and after washing them at 4 h postinfection as described above, coverslips were allowed to incubate for 48 h postinfection at 35°C with 7% CO₂ prior to fixation and DAPI staining.

Oxygen consumption assays.The oxygen consumption rate (OCR) of digitonin-permeabilized epimastigotes was measured using a high-resolution Oroboros Oxygraph-2k oxygraph (Oroboros Instruments GmbH, Innsbruck, Austria) with DatLab 4 software for data acquisition and analysis. The equipment was calibrated as reported by its manufacturer. Parasites from the cultures for wild-type, scrambled sgRNA, TcMPC1-KO, and TcMPC2-KO (1 × 10⁸ cells), pTREX-n/3×HA, TcMPC1-OE, and TcMPC2-OE (8.5 × 10⁷ cells) were washed with buffer A with glucose (BAG; 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 50 mM HEPES, pH 7.2, and 5.5 mM glucose). Then, cells were incubated at 28°C and 750 rpm in a 2-ml chamber containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH, pH 7.2, 1 mM MgCl_2, 2.5 mM K₂HPO₄, 0.2% BSA, 50 μM EGTA, and 5 mM mitochondrial substrate (succinate, pyruvate, or α-ketoglutarate, as indicated in the figure legends). Sequential additions of digitonin (25 μM), ADP (100 μM), oligomycin (1 μg ml⁻¹), and FCCP (1 μM) were performed during reading time. Inhibitors UK5099 (50 μM) and malonate (2 mM) were independently added into the chamber prior to reading in each experiment of inhibition using pyruvate as the mitochondrial substrate. OCR was calculated as the negative time derivative of O₂ concentration measured in the closed oxygraph chambers and expressed per milligram of protein. Data were recorded at 2-s intervals, and 10 data points were used to calculate the OCR slope plot through a polynomial fit with DatLab 4 software, as previously described (56). For comparison between OCRs of mutant cells and OCRs of control cells, normalized OCR was calculated from the normalization of OCR after an addition of ADP, oligomycin, or FCCP in relation to the basal OCR obtained right before ADP addition. A normalized OCR of 1 means that OCR after an addition is equal to the basal OCR; n independent biological experiments are indicated in the respective figure legends.

Mitochondrial calcium uptake assay.Mitochondrial Ca²⁺ uptake in permeabilized scrambled, TcMPC1-KO, and TcMPC2-KO epimastigotes was assayed by fluorescence of calcium green-5N probe at 28°C, as described previously (53), with the addition of 5 mM succinate or pyruvate to the reaction buffer (as indicated in the figures). Three independent experiments were performed.

Mitochondrial membrane potential assay.Mitochondrial membrane potential was assessed spectrofluorometrically using the indicator dye safranine O, as described previously (53, 57) with the following modification: the reaction buffer contained 5 mM pyruvate or succinate (as indicated in the figure legends). Additions of ADP (250 μM), CAT (1.5 μM), and FCCP (4 μM) were performed at different time points. The adenine nucleotide translocator inhibitor CAT was used in the membrane potential assay instead of oligomycin, because the basal fluorescence is only reached with CAT following the addition of ADP. Three independent experiments were performed.

α-Ketoglutarate dehydrogenase activity assay.The measurements of α-ketoglutarate dehydrogenase (α-KGDH) activity in control, TcMPC1-KO, and TcMPC2-KO epimastigotes were performed as described previously (58) with some modifications. Briefly, protein extracts from scrambled, TcMPC1-KO, and TcMPC2-KO epimastigotes were obtained by centrifugation of 10 ml of culture in exponential phase (1 × 10⁷ to 2 × 10⁷ cells ml⁻¹), washed with PBS, pH 7.4, at RT, and resuspended in 100 μl lysis buffer (0.1% Triton X-100, 50 mM K₂HPO₄ [pH 7.35] and 5 U ml⁻¹ Benzonase). Protein extracts were incubated for 10 min on ice before protein quantification with a BCA protein assay kit. The conversion of α-ketoglutarate, NAD⁺, and coenzyme A to succinyl-CoA, NADH, and CO₂ catalyzed by α-KGDH was measured in 96-well plates by following NADH fluorescence for 10 min at wavelengths of 340 and 460 nm for excitation and emission, respectively, using a Synergy H1 hybrid reader (BioTek, Winooski, VT). The α-KGDH activity assay was carried out in reaction buffer containing 50 mM K₂HPO₄ (pH 7.35), 1 mM MgCl₂, 0.2 mM thiamine pyrophosphate, 0.3 mM dithiothreitol, 100 μM EGTA, 50 μM coenzyme A-SH, 2 mM NAD⁺, 1 mM α-ketoglutarate, and 5 μg protein extract in a final volume of 260 μl, at RT. Blank wells containing lysis buffer instead of protein extracts were included in each assay, and their mean fluorescence was subtracted from that in the reaction wells. V_max values were used to normalize the α-KGDH activity from each cell line using control parasites as a reference sample. Three independent experiments were performed.

Statistical analyses.Values are expressed as means ± standard errors of the means (SEMs). Significant differences between treatments were compared using one-way and two-way analysis of variance (ANOVA) tests and the Kruskal-Wallis test, where indicated. Differences were considered statistically significant at P value of <0.05, and n refers to the number of experiments performed. All statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software, San Diego, CA).