Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the F o F 1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the F o F 1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨ m. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨ m. In totality, the data establish that the bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes

© 2020 Zoltner et al. Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite’s invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs’ cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death

The ATP/ADP carrier is dispensable for BSF <i>T</i>. <i>brucei</i> 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).</p

Metabolomic analysis of AAC DKO cells.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.</div

SCS DKO cells are viable <i>in vitro</i> 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.</p

SCS RNAi silencing results in growth phenotype and decreased ΔΨ_m 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).</p

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.</p

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 P m 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.</p

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.</p