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

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 [3H]-adenosine and [3H]-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 [3H]-diminazene, but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1

Comparative genomics of drug resistance in <i>Trypanosoma brucei rhodesiense</i>

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine

A Galleria mellonella infection model reveals double and triple antibiotic combination therapies with enhanced efficacy versus a multi-drug resistant strain of Pseudomonas aeruginosa.

The aim of this study was to compare the inhibitory effect of antibiotic combinations in vitro with efficacy in G. mellonella larvae in vivo to identify efficacious combinations that target P. aeruginosa. P. aeruginosa NCTC13437, a MDR strain resistant to -lactams and aminoglycosides, was used. Susceptibility to CTX, PIP, MER, AMK, LVX and CST alone, or in dual or triple combinations, was measured in vitro via a 24 h time-kill assay. In vitro results were then compared with the efficacy of the same dual or triple antibiotic combinations versus G. mellonella larvae infected with P. aeruginosa. G. mellonella haemolymph burden of P. aeruginosa was determined over 96 h post-infection and treatment with the most potent combination therapies. Many dual and triple combinations of antibiotics displayed synergistic inhibition of MDR P. aeruginosa in vitro. There was little correlation between combinations that were synergistic in vitro and those that showed enhanced efficacy in vivo versus infected G. mellonella larvae. The most potent dual and triple combinations in vivo were CTX + PIP and MER + PIP + AMK respectively. Fewer combinations were found to offer enhanced therapeutic benefit in vivo compared to in vitro. The therapeutic benefit arising from treatment with antibiotic combinations in vivo correlated with reduced larval burden of P. aeruginosa. This study has identified antibiotic combinations that merit further investigation for their clinical potential and has demonstrated the utility of using G. mellonella to screen for novel antibiotic treatments that demonstrate efficacy in vivo