Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei - PubMed (original) (raw)
doi: 10.1128/EC.2.5.1003-1008.2003.
Mhairi L Stewart, Federico Geiser, Reto Brun, Pascal Mäser, Lynsey J M Wallace, Richard J Burchmore, John C K Enyaru, Michael P Barrett, Ronald Kaminsky, Thomas Seebeck, Harry P de Koning
Affiliations
- PMID: 14555482
- PMCID: PMC219364
- DOI: 10.1128/EC.2.5.1003-1008.2003
Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei
Enock Matovu et al. Eukaryot Cell. 2003 Oct.
Abstract
Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.
Figures
FIG. 1.
Uptake of [3H]adenosine by the _tbat1_-null mutant. Uptake of 20 nM [3H]adenosine was inhibited by inosine (filled squares) with an IC50 of 1.1 ± 0.1 μM. Adenine failed to inhibit [3H]adenosine at concentrations as high as 100 μM when it was added alone (open squares). The inhibition induced by 1 mM adenine is attributable to a low-affinity inhibition of P1.
FIG. 2.
Uptake of [3H]pentamidine by the _tbat1_-null mutant. The uptake of 15 nM [3H]pentamidine was unaffected by as much as 1 mM adenosine (filled circles) but was inhibited to a maximum of 64% by propamidine (open squares), with an IC50 of 6.5 μM. When increasing amounts of unlabeled pentamidine (filled squares) were added, [3H]pentamidine uptake was inhibited in a biphasic manner (P < 0.0001 by the F test), with the high-affinity component (IC50 = 25.1 nM) contributing 62% of total [3H] pentamidine transport. The IC50 of the low-affinity component was 19.5 μM for this experiment.
FIG. 3.
In vitro sensitivities of bloodstream-form trypanosomes to arsenical trypanocides. (a) Wild-type T. b. brucei s427 parasites were incubated with or without 10 μM melarsen oxide in the presence of potential inhibitors. Traces: a, control (no arsenical); b, melarsen oxide only; c, melarsen oxide plus 4 mM adenosine; d, melarsen oxide plus 1 mM pentamidine; e, melarsen oxide plus 4 mM hypoxanthine. (b) Cells of the _tbat1_-null mutant were incubated with or without 10 μM cymelarsan. Traces: a, control (no arsenical); b, cymelarsan only; c, cymelarsan plus 0.1 μM pentamidine; d, cymelarsan plus 0.03 μM pentamidine; e, cymelarsan plus 0.01 μM pentamidine; f, cymelarsan plus 10 μM stilbamidine; g, cymelarsan plus 1 μM propamidine; h, cymelarsan plus 0.3 μM propamidine; i, cymelarsan plus 0.1 μM propamidine. (c) Cells of the _tbat1_-null mutant were incubated with or without 0.5 μM phenylarsine oxide. Traces: a, control (no arsenical); b, phenylarsine oxide only; c, phenylarsine oxide plus 10 mM adenosine; d, phenylarsine oxide plus 4 mM hypoxanthine; e, phenylarsine oxide plus 1 mM pentamidine; f, phenylarsine oxide plus 100 μM pentamidine; g, phenylarsine oxide plus 100 μM propamidine; h, phenylarsine oxide plus 100 μM stilbamidine. Arrow indicates time of phenylarsine oxide addition.
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