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Review
. 2015 Mar 9:6:32.
doi: 10.3389/fphar.2015.00032. eCollection 2015.

Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

Jane C Munday  1 , Luca Settimo  2 , Harry P de Koning  1
Affiliations
Review

Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

Jane C Munday et al. Front Pharmacol. .

Abstract

Drug resistance in pathogenic protozoa is very often caused by changes to the 'transportome' of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil(®)), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the "selectivity region" of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability.

Keywords: HAPT1; Trypanosoma brucei; aquaglyceroporin; aquaporin; drug resistance; drug transport; melarsoprol; pentamidine.

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Figures

FIGURE 1
FIGURE 1
Schematic of the documented AQP2 and AQP3 loci in lab-derived and field-isolated strains of Trypansoma brucei. Brown lines = SNPs in chimeras compared to WT; yellow lines = position of NSA/NPS loci from TbAQP2; pink lines = position of NPA/NPA loci from TbAQP3. (A) Locus found in wildtype strains (T. b. brucei Lister 427, 247, and TREU927; T. b. gambiense 386, STIB 930, and DAL972; and T. b. rhodesiense STIB900 (minor differences in TbAQP3 are not highlighted); (B) Locus with chimera TbAQP2-3(569-841) from lab-derived pentamidine resistant strain B48 (Baker et al., 2012); (C) Locus of chimera TbAQP2-3(376) from lab-derived melarsoprol resistant strain 247-Mr (Munday et al., 2014); (D) Locus in lab-derived melarsoprol resistant strains 386-Mr and STIB900-Mr; and lab-derived pentamidine resistant strain STIB900-PR (Munday et al., 2014), and in one T. b. gambiense K03048 allele (Graf et al., 2013); (E) Locus with chimera TbAQP2-3(814) found in field isolates from Mbuji-Masi locus in DRC and the other K03048 allele (Graf et al., 2013; Pyana Pati et al., 2014); (F) Locus with chimera TbAQP2-3(880) found in all T. b. gambiense field strains from Mbuji-Masi (Pyana Pati et al., 2014); (G) Locus with chimera TbAQP2-3(678-880) found in two old Congolese T. b. gambiense field strains, MBA and KEMLO (Pyana Pati et al., 2014) and (H) Chimera TbAQP2-3(617-658), without loss of AQP3, from four T. b. gambiense field strains isolated in Masi-Manimba (Pyana Pati et al., 2014).
FIGURE 2
FIGURE 2
Predicted binding of pentamidine and melarsoprol in complex with a single TbAQP2 subunit (green). (A) Binding of pentamidine (cyan carbon atoms). (B) Binding of melarsoprol (orange carbon atoms). Key polar interactions are shown for both (A,B). (C) Top extracellular view of the overlay of the docked binding poses of pentamidine (cyan carbon atoms) and melarsoprol (orange carbon atoms). The protein was modeled using MODELLER 9.14 (Sali and Blundell, 1993), using as a template the crystal structure of PfAQP (PDB code: 3c02) published by Newby et al. (2008). The sequence identity between TbAQP2 and the template was 33%. The images were created using PyMOL version 1.50.04 (Schrödinger). PyMOL was used to generate the biological units for the aquaglyceroporin from Plasmodium falciparum (generation of symmetry mates). Molecular docking was performed using FRED (McGann, 2012), using a multiconformer database generated using OMEGA. The docked poses were energy minimized using SZYBKI (version 1.7.0) allowing partial relaxation of the protein residues in the direct proximity to the ligand. FRED, OMEGA, and SZYBKI are software developed by OPENEYE (OpenEye Scientific Software: Santa Fe, NM, USA).
FIGURE 3
FIGURE 3
Extracellular top view of the tetrameric structures. (A) TbAQP2; the residues constituting the NSA, NPS, and IVLL motif are shown in space-filling models and the locations of Ile110, Leu258, and Leu 264 are indicated in one subunit. (B) TbAQP3; the residues constituting the two NPA and WGYR motif are shown in space-filling models and the locations of Trp102, Tyr250, and Arg256 are indicated in one subunit. (C) TbAQP2-3(569-841); the residues constituting the NSA, NPS, and IGYR motif are shown in space-filling models and the locations of Ile110, Tyr258, and Arg264 are indicated in one subunit. The images were created using PyMOL version 1.50.04 (Schrödinger). The proteins were modeled using MODELLER 9.14 (Sali and Blundell, 1993), using as a template the crystal structure of PfAQP (PDB code: 3c02; (Newby et al., 2008)). The sequence identity between the target and the template was 33, 36, and 34% for TbAQP2, TbAQP3, and the TbAQP2-3(569-841) chimera, respectively. The C-alpha atoms of chain A, B, C, and D of the tetramer template were restrained during homology modeling using MODELLER in order to reduce the number of interatomic distances that need to be calculated.
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