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Functional analysis of drug resistance‐associated mutations in the T rypanosoma brucei adenosine transporter 1 ( TbAT 1) and the proposal of a structural model for the protein
Author(s) -
Munday Jane C.,
Tagoe Daniel N. A.,
Eze Anthonius A.,
Krezdorn Jessica A. M.,
Rojas López Karla E.,
Alkhaldi Abdulsalam A. M.,
McDonald Fiona,
Still Jennifer,
Alzahrani Khalid J.,
Settimo Luca,
De Koning Harry P.
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12979
Subject(s) - biology , transporter , trypanosoma brucei , atp binding cassette transporter , allele , mutation , single nucleotide polymorphism , adenosine , genetics , pharmacology , microbiology and biotechnology , biochemistry , gene , genotype
Summary The T rypanosoma brucei aminopurine transporter P 2/ TbAT 1 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 TbAT 1 ‐ WT allele, although several combinations, and the deletion of the codon for residue F 316, resulted in highly significant impairment. These combinations of SNPs , and Δ F 316, also strongly impaired the uptake of [ 3 H ]‐adenosine and [ 3 H ]‐diminazene, identical to the tbat1 −/− control. The TbAT 1 protein model predicted that residues F 19, D 140 and F 316 interact with the substrate of the transporter. Mutation of D 140 to alanine resulted in an inactive transporter, whereas the mutation F 19A 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 TbAT 1.