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.

In Vitro and In Vivo Evaluation of 28DAP010, a Novel Diamidine for Treatment of Second-Stage African Sleeping Sickness

“…We observed a resistance factor (IC 50 P2-KO /IC 50 wild type ) of 16, which was similar to that for DB829 and diminazene in the 72-h alamarBlue assay (14). Recently another transporter, aquaporin 2 (AQP2), was identified which is also involved in drug uptake and the development of resistance to pentamidine and melarsoprol (40,43,44). However, cross-resistance seems to be limited to the two diamidines pentamidine and 28DAP010, as the IC 50 s of the two diamidines for different T. b. gambiense field isolates do not correlate (r 2 ϭ 0.019) (Fig.…”

“…2). A similar effect was observed between pentamidine and diminazene (44). Analysis of T. b. gambiense field isolates with reduced susceptibility to pentamidine did reveal rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss (44).…”

“…Analysis of T. b. gambiense field isolates with reduced susceptibility to pentamidine did reveal rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss (44). The mutant T. b. gambiense field isolates were less susceptible to pentamidine and melarsoprol but still susceptible to diminazene (44) and DB829 (23) and, as can be seen in Fig. 2, also to 28DAP010.…”

Gene mutations in parasitic diseases Part II: Parasite gene mutations

“…In 2013, aquaglyceroporin transporter (TbAQP2) was identified as another transporter involved in melarsoprol/pentamidine resistance. It was found that mutations in the gene encoding TbAQP2 significantly correlated with pentamidine and melarsoprol resistance in the clinical isolates [181] . On the other hand, isometamidium is a veterinary drug used against African trypanosomiasis, targeting the trypanosomes kinetoplast.…”

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species

“…Indeed, disruption of the AQP1 homolog in the Leishmania-related parasite Trypanosoma brucei (the etiological agent of human African trypanosomiasis), TbAQP2, generates melarsoprol (As-based drug)-resistant parasites, and laboratory-selected melarsoprol-resistant trypanosomes also showed a disrupted TbAQP2 (18). In fact, T. brucei gambiense field isolates with reduced susceptibility to melarsoprol harbor TbAQP2 mutations and gene loss (19). A similar functional correlation was demonstrated in L. mexicana, in which the heterologous expression of TbAQP2 increased the sensitivity of arsenicals Ͼ1,000-fold (20).…”