Fig 1.
Schematic and simplified representation of possible metabolic pathways related to cytosolic and mitochondrial ATP production.
Enzymes are: ACH, acetyl-CoA thioesterase; AOX, alternative oxidase; ASCT, acetate:succinate CoA-transferase, Gly-3-P DH, glycerol-3-phosphate dehydrogenase; KDH, α-ketoglutarate dehydrogenase; PK, pyruvate kinase; PDH, pyruvate dehydrogenase complex; SCS, succinyl-CoA synthetase; TDH, threonine dehydrogenase. Abbreviations: α-KG, α-ketoglutarate; ΔΨm, mitochondrial membrane potential; AAC, ATP/ADP carrier; CATR, carboxyatractyloside; OLM, oligomycin; TPMP, methyltriphenylphosphonium.
Fig 2.
The ATP/ADP carrier is dispensable for BSF T. brucei viability and for maintaining the ΔΨm.
(A) The strategy to generate AAC DKO involved replacement of both alleles with T7 RNA polymerase and tetracycline repressor linked to genes conferring neomycin and hygromycin resistance, respectively. (B) PCR verification for the elimination of all AAC alleles in AAC DKO cell line. The primers used are color-coded in (A). (C) Immunoblot analysis of AAC DKO cells using specific anti-AAC antibody. Immunodetection of mitochondrial hsp 70 served as a loading control. (D) Growth of AAC DKO cells compared to wild-type BSF 427 in HMI-11 measured for 8 days. (E) Growth of AAC DKO cells compared to wild-type BSF 427 in CMM medium measured for 7 days. (F) The survival rate of 5 female BALB/c mice which were intraperitoneally infected with AAC DKO and wild-type BSF 427 parasites. The infected mice were monitored for 6 days. (G) Flow cytometry analysis of TMRE-stained AAC DKO and BSF 427 cells grown in HMI-11 or CMM medium to measure ΔΨm. The addition of FCCP served as a control for ΔΨm depolarization (+FCCP). (means ± s.d., n = 6). (H) Flow cytometry analysis of TMRE-stained AAC DKO and BSF 427 cells grown in HMI-11 medium and treated with 250 ng/ml of oligomycin (+OLM) for 24 hours before the analysis. (means ± s.d., n = 6).
Fig 3.
In the absence of AAC, the cells are unable to import cytosolic ATP to the mitochondrial matrix.
(A) Mitochondrial membrane polarization detected using Safranine O dye in digitonin-permeabilized BSF 427 cells in the presence of ATP. Carboxyatractyloside (CATR), the AAC inhibitor was added before the ATP (red line) as a control for no membrane polarization due to the inability to import ATP into the mitochondrial matrix. Oligomycin (OLM) was added after the CATR to induce depolarization. SF6847, an uncoupler, was added to test any further depolarization. ATP, CATR, OLM and SF 6847 were added where indicated. (B) Mitochondrial membrane polarization detected using Safranine O dye in digitonin-permeabilized AAC DKO and AAC DKO Addback cells in the presence of ATP. CATR, OLM and SF 6847 were added where indicated. The inset shows western blot analysis of BSF 427, AAC DKO Addback cells grown in the presence or absence of tetracycline, probed with anti-v5 monoclonal antibody, that recognizes the v5 epitope attached to the 3´end of the AAC gene, and anti-mt Hsp70 antibody as a loading control. (C) Subcellular localization of v5-tagged luciferase without (luc_cyto) or with mitochondrial localization signal (luc_mito) endogenously expressed in BSF 427 and AAC DKO cells was determined in whole cell lysates and in the corresponding cytosolic and organellar fractions separated by digitonin extraction. Purified fractions were analyzed by Western blotting with the following antibodies: anti-v5, anti-mt Hsp70 (mitochondrial marker), and anti-adenosine phosphoribosyltransferase (APRT) (cytosolic marker). The relevant sizes of the protein marker are indicated on the left. (D) Representative data of basal (first peak) and glucose-induced (second peak) levels of bioluminescence detected by a plate reader in the cytosol of BSF 427_luc_cyto (left panel) and AAC DKO_luc_cyto (right panel) using 25 μM luciferin. (E) Quantification of changes in ATP levels upon 5 mM glucose addition in BSF 427_Luc_cyto and AAC DKO_luc_cyto. Box and whiskers plots, n = 7–10, *** P < 0.001. (F) Representative data of basal (first peak) and glucose-induced (second peak) bioluminescence levels detected by a plate reader in the mitochondrial matrix of BSF 427_luc_mito (left pane) and AAC DKO_luc_mito (right panel) using 25 μM luciferin. (G) Quantification of changes in ATP levels upon 5 mM glucose addition in BSF 427_Luc_mito and AAC DKO_luc_mito. Box and whiskers plots, n = 8–11, *** P < 0.001.
Fig 4.
AAC DKO cells are more sensitive to the treatment by TPMP, an inhibitor of α-ketoglutarate dehydrogenase.
(A) Sensitivity of BSF 427, AAC DKO, AAC DKO_addback to TPMP estimated by Alamar blue cell viability assay. (B) AAC DKO/ASCT RNAi noninduced (NON) and cells induced for 4 days (D4) to TPMP estimated by resazurine cell-viability assay. The dose-response curves were calculated using GraphPad Prism 8.0 software. The calculated EC50 values are shown in graphs and are expressed in μM. (C) Western blot analysis of BSF 427, AAC DKO and AACDKO/ASCT RNAi cells uninduced and induced for 2 and 4 days using anti-ASCT antibody. *-non-specific band serving as a loading control.
Fig 5.
SCS DKO cells are viable in vitro but exert lower virulence in animal model.
(A) The strategy to generate SCS DKO involved replacement of both alleles with resistance genes conferring neomycin and hygromycin resistance. (B) PCR verification for the elimination of both SCS alleles in SCS DKO cell line. (C) Immunoblot analysis of SCS DKO cells using specific anti-SCS antibody. Immunodetection of cytosolic APRT served as a loading control. (D) Subcellular localization of SCS using BSF 427 cells. WCL, whole cell lysate; Cyt, cytosol; Mito, mitochondrial; insol, insoluble; sol, soluble. (E) Enzymatic activity of SCS measured in mitochondrial lysates extracted from BSF 427, AAC DKO and SCS DKO cells. (F) Growth of AAC DKO cells compared to wild-type BSF 427 in HMI-11 and CMM medium measured for at least 7 days. (G) The survival rate of 7 female BALB/c mice which were intraperitoneally infected with SCS DKO and wild-type BSF 427 parasites. The infected mice were monitored for 14 days. (H) The survival rate of 7 female BALB/c mice which were intraperitoneally infected with SCS DKO Addback and wild-type BSF 427 parasites. The SCS DKO Addback infected mice were supplied with water containing doxycycline to induced expression of the addback SCS copy. The mice were monitored for 6 days. (I) Immunoblot analysis of BSF 427 and SCS cDKO cell line inducibly expressing v5-tagged SCS using specific anti-SCS antibody. Immunodetection of mitochondrial hsp70 served as a loading control. (J) The survival rate of 7 female BALB/c mice which were intraperitoneally infected with BSF 427 and SCS DKO_addback parasites.
Fig 6.
SCS DKO parasites do not excretes acetate.
Proton (1H) NMR analyses of end-products excreted from the metabolism of 13C-enriched glucose. BSF 427 (A), SCS DKO (B) and AAC DKO (C) trypanosomes were incubated for 2.5 hours in PBS containing 4 mM [U-13C]-glucose in combination with threonine (+Thr) or α-ketoglutarate (+α-KG) before analysis of the spent medium by 1H-NMR spectrometry. The amounts of each end-product excreted are documented in S3 Table. Abbreviations: Ac, acetate; Al, alanine; L, lactate; Py, pyruvate; S, succinate.
Fig 7.
SCS DKO parasites have decreased mitochondrial ATP content, but are capable of ATP import and ATP hydrolysis.
(A) Subcellular localization of V5-tagged luciferase with mitochondrial localization signal (luc_mito) endogenously expressed in SCS DKO cells was determined in whole cell lysates and in the corresponding cytosolic and organellar fractions separated by digitonin extraction. Purified fractions were analyzed by Western blotting with the following antibodies: anti-v5, anti-mt Hsp70 (mitochondrial marker), and anti-adenosine phosphoribosyltransferase (APRT) (cytosolic marker). The relevant sizes of the protein marker are indicated on the left. (B) Immunoblot of V5-tagged luciferase expressed in BSF 427_luc_mito, AAC DKO_luc_mito and SCS DKO_luc_mito cells using antibodies against V5 tag. Antibody against subunit p18 of FoF1 ATP synthase was used as a loading control. (C) The quantification analyses of luciferase expression in all three cell lines by densitometry. The bars represent relative protein amounts of luciferase expression in AAC DKO and SCS DKO cells compared to luciferase expression in BSF 427. (means ± s.d., n = 6–7). (D) Representative data of ATP measurements performed in living BSF 427_luc_mito, AAC DKO_luc_mito and SCS DKO_luc_mito cells using 25 μM luciferin. (E) Quantification of the luminescence measurement detected in BSF 427_luc_mito, AAC DKO_luc_mito, SCS DKO_luc_mito. Data shown in the bars are derived from experiments of which representative graphs are shown in panel D (means ± s.d., n = 5, Student´s unpaired t-test, *P < 0.05, ** P < 0.005). (F) Flow cytometry analysis of TMRE-stained SCS DKO and BSF 427 cells grown in HMI-11 or CMM medium to measure ΔΨm. The addition of FCCP served as a control for ΔΨm depolarization (+FCCP). (means ± s.d., n = 6). (G, H) Mitochondrial membrane polarization detected using Safranine O dye in digitonin-permeabilized BSF 427 cells (black/grey lines) and SCS DKO (light and dark red) in the presence of ATP. ATP, CATR, OLM and SF 6847 were added where indicated.
Fig 8.
SCS DKO cells are more sensitive to CATR, an inhibitor of AAC.
(A) Sensitivity of BSF 427, SCS DKO, ASCT DKO to carboxyatractyloside (CATR) estimated by Alamar Blue cell-viability assay. The dose-response curves were calculated using GraphPad Prism 8.0 software. The calculated EC50 values are shown in graphs and are expressed in mM. (B) Sensitivity of BSF 427 and SCS DKO, ASCT DKO to bongkrekic acid estimated as in (A). (C) Sensitivity of BSF 427, SCS cDKO noninduced (-tet) and 4-days induced (+tet) cells to carboxyatractyloside (CATR) estimated as in (A). (D) Immunoblot of SCS cDKO noninduced (-tet) and 2-days induced (+tet) cells using SCS antibody. Immunodetection of mitochondrial hsp70 served as a loading control.
Fig 9.
SCS RNAi silencing results in growth phenotype and decreased ΔΨm in CMM_glc and CMM_gly medium.
(A) Growth of BSF 427, AAC DKO and SCS DKO cells in CMM_glc and CMM_gly medium. (B) Western blot analysis of whole cell lysates of SCS RNAi non induced and induced (+tet) cells grown in HMI-11, CMM_glc and CMM_gly using antibodies against the SCS protein. The immunoblot probed with anti-mitochondrial hsp70 antibody served as loading controls. Glc, glucose; gly, glycerol. (C) Growth of SCS RNAi noni nduced (non) and tetracycline induced (IND) cells measured for 8 days in HMI- 11 (left), CMM_glc (middle) and CMM_gly (right). Glc, glucose; gly, glycerol. (D) Flow cytometry analysis of TMRE-stained SCS RNAi noninduced and induced cells grown in HMI- 11 (right), CMM_glc (middle) and CMM_gly (left). (means ± s.d., n = 3–9).
Fig 10.
Schematic visualization of AAC and SCS activities interplay in BSF 427 (A), AAC DKO (B) and SCS DKO (C) grown in HMI-11 and BSF 427 cultured in CMM_gly medium (D).
AAC, ATP/ADP carrier; SCS, succinyl-CoA synthetase.