Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol

Abstract

The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.

Figure 1. Schematic view of the TbAQP2/TbAQP3 locus on chromosome 10.

A) Reference locus of T. b. brucei TREU927, T. b. gambiense STIB 930 and T. b. gambiense DAL972 (minor differences in TbAQP3 are not highlighted). B) Chimera of TbAQP2 and TbAQP3 as described by Baker et al. (2012) for the in vitro selected, pentamidine-resistant T. b. brucei line B48. C) Chimera of TbAQP2 and TbAQP3 plus loss of TbAQP3 in T. b. gambiense 40 AT, 45 BT, 130 BT, 349 BT, and 349 AT, and in one K03048 allele. D) Deletion of the TbAQP2 ORF in the other T. b. gambiense K03048 allele. E) TbAQP2 polymorphisms (C474A, G475A, C477T, T480C) in several T. b. rhodesiense and T. b. brucei isolates from East Africa (STIB 900, STIB 950, STIB 940, and STIB 871).

Figure 2. In vitro drug sensitivities.

50% inhibitory concentrations (IC₅₀) as determined with the Alamar blue assay. Susceptibility to pentamidine correlates with that to melarsoprol but not diminazene. TbAT1 and TbAQP2 genotypes are indicated.