Authors

N Wiedemar²; M Zwyer²; M Zoltner¹; F E Graf²; E Ndomba²; C Kunz Renggli²; M Cal²; R S Schmidt²; T Wenzler²; M C Field¹; P Mäser²;  ¹ Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, UK;  ² Swiss Tropical and Public Health Institute, Basel, Switzerland, Switzerland

Discussion

Suramin is the drug of choice to treat the first stage of the acute form of sleeping sickness, caused by T. brucei rhodesiense. Despite its use for a century, knowledge about the drug is still limited. Investigating drug resistance mechanisms can help to identify the target, transport and mode of action of a drug. For suramin, we observed the emergence of a high resistance in a T. b. rhodesiense strain (STIB900) after exposure to the drug for only few days. Here we investigate the genetic and biochemical mechanisms behind this phenomenon.

A fresh STIB900 clone was exposed to suramin in vitro and four independently selected, resistant derivatives were generated. They were phenotypically characterized and mRNA sequencing was carried out to find differentially expressed genes between suramin sensitive and resistant cells. The identified candidate gene was validated by reverse genetic in situ gene replacement. And the effect on endocytosis of different substrates was investigated by FACS and fluorescence microscopy.

Suramin resistant derivatives were obtained after 6 days of selection and showed a resistance factor around 100-fold compared to the sensitive parent clone. They were cross-resistant to trypan blue and had a mild growth defect. Gene expression analysis revealed a switch to the same variant surface glycoprotein (VSG), termed VSG^Sur, in all the resistant derivatives. No other genes were differentially expressed between resistant and sensitive cells. We then introduced VSG⁹⁰⁰, which was expressed in the sensitive parent clone, into the active expression site of one resistant derivative, and thereby replaced VSG^Sur. Through this manipulation, the cells completely lost their resistance. Complementary, the introduction of VSG^Sur into the active expression site of T. b. brucei 2T1 cells led to suramin resistance. Upon expression of VSG^Sur, uptake of trypan blue was reduced, and uptake of low density lipoprotein and transferrin were highly reduced.

Here we describe a previously unknown VSG (VSG^Sur), which causes a strong suramin resistance. The expression of VSG^Sur not only confers resistance, but also alters the uptake of a number of substrates including nutrients, thus supposedly has a major impact on the biology of the cells.