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

Abstract

31Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to 32 pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively 33 selected for increased pore size from a common ancestor aquaporin. We demonstrate that 34 TbAQP2's unique architecture permits pentamidine permeation through its central pore and 35 show how specific mutations in highly conserved motifs affect drug permeation. Introduction 36 of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. 37 Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically 38 favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally 39 strictly impermeable for ionic species. We also identify the structural determinants that make 40 pentamidine a permeant but exclude most other diamidine drugs. Our results have wide-41 ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. 42Moreover, these new insights in aquaporin permeation may allow the pharmacological 43 exploitation of other members of this ubiquitous gene family. 44 45 Keywords: 46 Drug transport / Aquaporin / evolution of membrane proteins / Trypanosoma brucei / 47 109 endocytosis rate and the rate of pentamidine uptake. Our results unequivocally show that 110 pentamidine permeates directly through the central pore of TbAQP2 and that uptake is 111 dependent on the microbial membrane potential. Having identified the essential 112 characteristics that allow the transport of large, flexible molecules through TbAQP2, this 113 should now allow the evaluation of aquaporins in other species for similar adaptations.114 7 Results 115 116 12. Investigation of the structural determinants of AQP2 for pentamidine transport 117 1.1. Positive selection for pore size 118 In T. brucei, the AQP2 and AQP3 genes are arranged as a tandem pair on chromosome 10 119 and have 74% amino acid identity. Whereas TbAQP2 clearly mediates pentamidine uptake, 120 TbAQP3 does not (Baker et al, 2012; Munday et al, 2014), nor do various chimeric AQP2/3 121 rearrangements that give rise to pentamidine resistance (Munday et al, 2014; Graf et al, 122 2015). To investigate the origin of the AQP2 gene, a phylogenetic analysis of AQPs in 123 African trypanosomes was performed. The number of aquaporin genes varies: there is a 124 single aquaporin in T. vivax and T. congolense, two in T. suis and three in T. brucei and its 125 derivatives (Supplemental Fig. 1A). The most probable tree (Supplemental Fig. 1B) is 126 consistent with the evolutionary history of the four species (Hutchinson & Gibson, 2015) and 127 indicates AQP1 as the ancestral AQP present in all trypanosome species. A duplication 128 occurred in the common ancestor of T. suis and T. brucei after divergence from T. 129 congolense and a further duplication, to form AQP2 and AQP3, in the ancestor of T. 130 brucei after divergence from T. suis. Multiple alignment (Supplemental Fig. 1A) shows that 131 the classical NPA/NPA a...

The Ionophores CCCP and Gramicidin but Not Nigericin Inhibit Trypanosoma brucei Aquaglyceroporins at Neutral pH

“…Again, we observed no change in the light scattering signal ( Figure 4 c). Since nigericin, like CCCP, has been linked to inhibition of pentamidine uptake in trypanosomes [ 2 , 22 ], we tested if nigericin affected TbAQP2 functionality in connection to pentamidine. We showed before that pentamidine acts as a direct inhibitor of TbAQP2 glycerol permeability [ 17 ].…”

“…The localization of TbAQP2 in the trypanosomal flagellar pocket is of particular interest due to the exceptionally high membrane turnover rates [ 18 , 19 , 20 ]. It is clear that pentamidine acts as a potent inhibitor of the glycerol permeability of TbAQP2 but not TbAQP3 in the yeast-based system [ 17 ] as well as of TbAQP2 in trypanosomes [ 22 ]. Consistent with this, pentamidine uptake in trypanosomes was found to be mediated by TbAQP2 and not TbAQP3 [ 5 ].…”

“…In any case, as a cationic molecule, uptake of pentamidine into trypanosomes should benefit from the generally negative membrane potential. It has been suggested that pentamidine uptake further depends on the proton motive force established by three plasma membrane H + -ATPases [ 21 ], and treatment with ionophores indeed led to decreased pentamidine uptake [ 2 , 22 ]. Ionophores are molecules that facilitate transmembrane transport of certain ions including protons (protonophores) either by shuttling the ion across the membrane in a bound form to the ionophore, or by forming ion-permeable channels in the membrane.…”

“…As a consequence, transmembrane ion or pH gradients will break down eliciting a decoupling effect. As a caveat, at least one of the protonophores used in the pentamidine experiments, carbonyl cyanide m -chlorophenyl hydrazone (CCCP), appeared to directly affect TbAQP2 functionality [ 22 ]. This prompted us to test for direct inhibition of TbAQP2 by common ionophores using conditions that exclude indirect effects via the disturbance of transmembrane charge distributions.…”

The Drugs of Sleeping Sickness: Their Mechanisms of Action and Resistance, and a Brief History

“…This allowed docking of the stretched-out pentamidine in a low-energy conformation inside the pore of a model of TbAQP2 [62]. Much work has subsequently been done to supply definitive proof of either model, and this has definitively come down on the side of uptake through the TbAQP2 pore [63].…”