Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates

Abstract

Aquaglyceroporin-2 is a known determinant of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2-AQP3 tandem locus was described from melarsoprol-pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2-aqp3 null T. b. brucei does not. This proves that AQP2-AQP3 chimerization is the cause of melarsoprol-pentamidine cross-resistance in the T. b. gambiense isolates.

Keywords: Aquaporin; Drug resistance; Human African trypanosomiasis; Melarsoprol; Pentamidine; Reverse genetics; Sleeping sickness; Trypanosoma brucei gambiense.

Graphical abstract

Fig. 1

Introduction of AQP2 into mutant T. b. gambiense. In vitro drug sensitivity of bloodstream-form T. b. gambiense 40AT (grey) transfected with AQP2 (black) or dysfunctional AQP2 (ctrl, dark grey). Error bars are standard errors of the mean. n = 6 independent experiments, each in duplicate. Small letters indicate significance groups as determined by one-way ANOVA and Tukey's post test using GraphPad Prism 5.0.

Fig. 2

Expression of the AQP2/3₍₈₁₄₎ chimera in T. b. brucei. In vitro drug sensitivity of bloodstream-form T. b. brucei 2T1 aqp2–aqp3 double null mutants (A) and parental 2T1 cells (B) transfected with a tetracycline (tet) inducible AQP2/3₍₈₁₄₎ chimera. Dark bars, tet (1 µg/ml) was added 24 h prior to the drug assay. Error bars are standard error of the mean. n = 4–5 independent experiments, each in duplicate. (C) Western blot with anti-GFP antibody demonstrating inducible expression of GFP-tagged AQP2/3₍₈₁₄₎ (Coo, Coomassie stain). The GFP-AQP2/3₍₈₁₄₎ fusion proteins ran below their predicted molecular mass (approximately 60 kDa), which often applies for proteins with many transmembrane domains. The lower of the inducible bands in the blot on the right may represent unprocessed (e.g. unglyosylated) GFP-AQP2/3.