Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei

Abstract

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/ glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmicoriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei

“…This fails to support a model in which pentamidine binds and is then internalized by endocytosis: the inhibitors do not show resistance in the tbaqp2/tbaqp3 null line, whereas substrates do. The lack of any correlation would be especially problematic for a model of ‘passive’ pentamidine endocytosis, where the drug merely piggy-backs on TbAQP2 as it is internalised in its regular turnover schedule (t 1/2 >4 h [ Quintana et al, 2020 ]), without inducing any conformation changes in the receptor protein. The caveat inherent to using the RF instead of rate of transport is that it cannot be excluded that some of the test compounds are AQP2 substrates yet predominantly taken up by transporters other than TbAQP2, and hence show a low RF.…”

“…The experimental V max for HAPT-mediated pentamidine uptake in T. brucei BSF and procyclics ( de Koning, 2001a ) can be expressed as 9.5 × 10 5 and 8.5 × 10 6 molecules/cell/h, respectively; given a 1:1 stoichiometry for AQP2:pentamidine the endocytosis model would require the internalisation and recycling of as many units of TbAQP2 and this seems unlikely, especially in procyclic cells, as even in BSF the half-life time for TbAQP2 turnover is >4 hr ( Quintana et al, 2020 ) and procyclic cells have a lower endocytosis rate and cannot easily internalise the aquaporins spread over the cell surface, as discussed above. Given the observed rate of uptake and turnover rate, this would require the presence of ~4 × 10 6 TbAQP2 units per BSF cell in the flagellar pocket.…”

“…Independence from endocytosis was investigated by employing the tetracycline-inducible CRK12 RNAi cell line previously described to give a highly reproducible and progressive endocytosis defect in T. brucei ( Monnerat et al, 2013 ), with the aim to distinguish between uptake via endocytosis and transporters, as current evidence suggests that in T. brucei all endocytosis, taking place exclusively in the flagellar pocket, is clathrin-dependent and AP-2 independent ( Morgan et al, 2002 ; Allen et al, 2003 ). This means that the endocytotic mechanisms of TbAQP2 and suramin receptor ISG75, which are both directed to the lysosome after ubiquitylation ( Quintana et al, 2020 ; Zoltner et al, 2015 ), are likely to be similar enough for a direct comparison. Twelve hours after CRK12 RNAi induction pentamidine transport was not significantly reduced although uptake of [ 3 H]-suramin, which is accumulated by endocytosis through the T. brucei flagellar pocket ( Zoltner et al, 2016 ), was reduced by 33% (p=0.0027), indicating successful timing of the experiment to the early stage of endocytosis slow-down.…”

“…We also show that pentamidine, at approximately its half-maximal occupancy concentration, did not influence the half-life time of TbAQP2 turnover, as is often the case in receptor-mediated endocytosis. Nor should TbAQP2, which exists as a rigid tetramer of tetramers in T. brucei bloodstream forms ( Quintana et al, 2020 ), be able to undergo the type of conformational change necessary to signal receptor occupancy and internalisation as observed in well-documented examples of ligand-triggered internalisation of transporters (e.g. Gournas et al, 2010 ; Gournas et al, 2017 ; Ghaddar et al, 2014 ; Keener and Babst, 2013 ).…”

“…Cells were washed with ice-cold PBS, then resuspended in 1 × SDS sample buffer (Thermo) and incubated at 70°C for 10 min. Proteins were subjected to electrophoresis using 4–12% precast acrylamide Bis-Tris gels (Thermo) and transferred to polyvinylidene difluoride (PVDF; Sigma-Aldrich) membranes with a iBlot2 system (Thermo) at 23 V for 6 min, exactly as described ( Quintana et al, 2020 ), blocking non-specific binding with 5% (w/v) bovine serum albumin (BSA; Sigma) in Tris-buffered saline (pH 7.4) with 0.2% (v/v) Tween-20 (TBST) and using rat anti-HA IgG 1 (Sigma) or anti-mouse β-tubulin (clone KMX-1; Millipore) at 1:5000 or 1:10,000 dilution in TBST, respectively. Membranes were washed five times with TBST and then incubated in TBST/1% BSA with the appropriate horseradish peroxidase (HRP)-coupled secondary antibody (Sigma), at 1: 10,000.…”