Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei adenosine transporter 1 (TbAT1) and the proposal of a structural model for the protein.

Abstract

The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

• Authors: Jane C Munday, Daniel N A Tagoe, Anthonius A Eze, Jessica A M Krezdorn, Karla E Rojas López, Abdulsalam A M Alkhaldi, Fiona McDonald, Jennifer Still, Khalid J Alzahrani, Luca Settimo, Harry P De Koning
• Journal: Molecular microbiology (Mol Microbiol) Vol. 96 Issue 4 Pg. 887-900 (May 2015) ISSN: 1365-2958 [Electronic] England
• PMID: 25708978 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Chemical References

• Nucleoside Transport Proteins
• Trypanocidal Agents
• adenosine transporter
• Pentamidine
• Diminazene
• Melarsoprol

Topics

• Alleles
• Diminazene (pharmacology)
• Drug Resistance, Multiple (genetics)
• Kinetics
• Melarsoprol (pharmacology)
• Models, Molecular
• Mutation
• Nucleoside Transport Proteins (chemistry, genetics, metabolism)
• Parasitic Sensitivity Tests
• Pentamidine (pharmacology)
• Polymorphism, Single Nucleotide
• Protein Interaction Domains and Motifs
• Trypanocidal Agents (pharmacology)
• Trypanosoma brucei brucei (chemistry, drug effects, genetics)