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

Abstract

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.

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei

“…Samples from the culture taken at this time point showed an increased abundance of cells with swelling characteristic of endocytosis defects, although this was hard to quantify as a minority of cells were affected, and to various degrees, as the 12 hr time point was deliberately taken at as early a point as possible, so as not to affect cell viability ( Figure 5—figure supplement 1 ) or cause excessive cellular pathology. We thus performed parallel uptake experiments with [ 3 H]-pentamidine and [ 3 H]-suramin, with suramin acting as positive control as it is known to enter T. brucei bloodstream forms through endocytosis after binding to surface protein ISG75 ( Zoltner et al, 2016 ; Zoltner et al, 2020 ). After 12 hr of CRK12 RNAi induction, pentamidine uptake was not significantly less than in the T. brucei 2T1 parental cells, whereas uptake of [ 3 H]-suramin was (p=0.019, n = 5; Figure 5B,C ).…”

Evolving Differentiation in African Trypanosomes

“…Moreover, these parasites could establish and sustain infection in the tsetse fly. In a second report, analysis of trypanosome responses to suramin in vitro indicates overall impairment in cellular metabolism with decreased levels of cellular ATP and induction of proteins associated with stumpy differentiation, including PAD1, PAD2, PIP39, and NRKA/B [57]. Many of the proteins upregulated in suramin-treated cells are involved in mitochondrial activity and the Krebs cycle, indicating activation of metabolic pathways similar to the stumpy phenotype, albeit in this case, likely arising from an ultimately fruitless attempt to generate ATP.…”

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda

“…Besides inhibition of glycolytic process, suramin reduce the overall cellular ATP levels and partially activates mitochondrial Krebs’ cycle ( Zoltner et al, 2020 ). In our analysis, most of the proteins associated with energy in suramin resistant strain are mitochondrial related.…”

“…As such, the upregulation of ISG75 in the suramin resistant isolate is an indication that this parasite isolate can sufficiently take up suramin. The ISG75 expression level correlate with suramin accumulation in the parasite ( Zoltner et al, 2020 ). We thus hypothesize that, 1) this parasite isolate may remain resistant to suramin due to inadequate expression of supporting internal lysosomal components required for the downstream effectiveness of drug action or 2) the possibility of the presence of mutation in ISG75 expressed by suramin resistance isolate that interfere with suramin binding efficiency hence lowering drug uptake cannot be underestimated.…”

“…The enrichment and overexpression of mitochondrial related proteins and/or enzymes in the suramin resistant isolate BSF indicate that this parasite isolate partially activates and utilizes mitochondrial ATP-generating activity. Over-expressed proteins in suramin resistant isolate are altered by suramin in the PC parasites compared to BSF trypanosomes ( Zoltner et al, 2020 ). As expected, the expression of some of the parasite proteins associated with suramin sensitivity were upregulated in the suramin sensitive isolate.…”