Abstract
Suramin was introduced into the clinic a century ago and is still used to treat the first stage of acute human sleeping sickness. Due to its size and sixfold negative charge, uptake is mediated through endocytosis and the suramin receptor in trypanosomes is thought to be the invariant surface glycoprotein 75 (ISG75). Nevertheless, we recently identified a variant surface glycoprotein (VSGSur) that confers strong in vitro resistance to suramin in a Trypanosoma brucei rhodesiense line. In this study, we introduced VSGSur into the active bloodstream expression site of a T. b. brucei line. This caused suramin resistance and cross resistance to trypan blue. We quantified the endocytosis of different substrates by flow cytometry and showed that the expression of VSGSur strongly impairs the uptake of low‐density lipoprotein (LDL) and transferrin, both imported by receptor‐mediated endocytosis. However, bulk endocytosis and endocytosis of the trypanolytic factor were not affected, and the VSGSur‐expressors did not exhibit a growth phenotype in the absence of suramin. Knockdown of ISG75 was synergistic with VSGSur expression, indicating that these two proteins are mediating distinct suramin resistance pathways. In conclusion, VSGSur causes suramin resistance in T. brucei bloodstream forms by decreasing specific, receptor‐mediated endocytosis pathways.
Highlights
Sleeping sickness, transmitted by the tsetse fly, is caused by two subspecies of the protozoan parasite Trypanosoma brucei: T. b. gambiense typically establishes a chronic infection, Abbreviations: ISG75, invariant surface glycoprotein 75; LDL, low‐density lipoprotein; VSG, variant surface glycoprotein.whereas T. b. rhodesiense causes a rather acute form of the disease
We quantified the endocytosis of different substrates by flow cytometry and showed that the expression of VSGSur strongly impairs the uptake of low‐density lipoprotein (LDL) and transferrin, both imported by receptor‐mediated endocytosis
We investigate the effects of enforced VSGSur expression in a suramin‐sensitive T. b. brucei line, aiming to uncover the molecular mechanisms underlying the link between VSGSur expression and suramin resistance
Summary
Sleeping sickness, transmitted by the tsetse fly, is caused by two subspecies of the protozoan parasite Trypanosoma brucei: T. b. gambiense typically establishes a chronic infection, Abbreviations: ISG75, invariant surface glycoprotein 75; LDL, low‐density lipoprotein; VSG, variant surface glycoprotein. Invariant surface glycoprotein 75 (ISG75) was proposed to act as the suramin receptor, since its knock‐down led to a decrease in suramin sensitivity.[6] Suramin shows a high plasma protein binding with approximately 70% bound to albumin, 15% to low‐density lipoprotein (LDL) and 15% free.[5] Based on findings that trypanosomes took up >20‐fold more suramin in the presence of LDL than in the presence of plasma, whereas the binding and uptake of LDL were up to 70% reduced in the presence of high micromolar concentrations of suramin,[5] suramin was proposed to be taken up in complex with LDL. The VSGSur expressing parasites showed an increase in IC50 of almost 100‐fold for suramin, and cross resistance to trypan blue, a trypanocidal dye related to suramin. We investigate the effects of enforced VSGSur expression in a suramin‐sensitive T. b. brucei line, aiming to uncover the molecular mechanisms underlying the link between VSGSur expression and suramin resistance
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