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Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei
Biochimica et Biophysica Acta 1859 (2017) 679–685
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Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei Sabrina Marsiccobetre a, Alexis Rodríguez-Acosta a, Florian Lang b, Katherine Figarella a,⁎, Néstor L. Uzcátegui a,b,⁎ a b
Laboratory of Immunochemistry and Ultrastructure, Institute of Anatomy, Central University of Venezuela, Caracas, Venezuela Department of Physiology I, University of Tubingen, Tubingen, Germany
a r t i c l e
i n f o
Article history: Received 5 October 2016 Received in revised form 3 January 2017 Accepted 7 January 2017 Available online 10 January 2017 Keywords: aquaglyceroporins boric acid Trypanosoma brucei heterologous expression
a b s t r a c t The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Diseases caused by members of the Trypanosomatidae family depict a relevant source of mortality and morbidity worldwide and especially in developing countries [1–3]. One of these diseases is the human African trypanosomiasis (HAT), or sleeping sickness, caused by two sub species of Trypanosoma brucei (T. b. gambiense and T. b. rhodesiense), which threatens millions of people in 36 countries in the sub-Saharan region and has a mortality rate nearly 100%, if it is not treated [4]. There are few drugs for the treatment of HAT, and unfortunately, most of them are highly toxic. Pentamidine and Suramin produce severe side effects and are ineffective in the chronic phase. The EflornithineNifurtimox combination is an acceptable treatment for the chronic stage of HAT caused by T. b. gambiense [5]. Meanwhile, Melarsoprol (Arsenal®, Aventis), a highly toxic arsenic derivative, is the only effective therapy against the chronic stage in patients infected with T. b. rhodesiense. Nevertheless, near 10% of patients treated with this drug develop a severe encephalopathy, which is fatal in half of the ⁎ Corresponding authors at: Universidad Central de Venezuela. Paseo Los Ilustres, Ciudad Universitaria. Instituto Anatómico. Caracas 1040, Venezuela. E-mail addresses: kfi[email protected] (K. Figarella), [email protected] (N.L. Uzcátegui).
http://dx.doi.org/10.1016/j.bbamem.2017.01.011 0005-2736/© 2017 Elsevier B.V. All rights reserved.
cases. Moreover, treatment failure of these drugs due to resistance is common [6]. Development of new more effective and safe trypanocidal compounds is urgently needed. Boron-containing molecules seem to be an interesting option, as they are effective against a wide range of infectious diseases [7]. The drugs for Neglected Diseases initiative (DNDi) organization, Anacor Pharmaceuticals, and their partners have developed several promising boron-containing molecules, the so-called oxaboroles. Several of them are highly active against HAT chronic phase in animal models. One of the oxaboroles, SCYX-7158, has already completed phase I clinical trials (DNDi). However, the action mechanisms of boron containing molecules are unknown [8]. Down regulation of membrane proteins responsible for drug transport into the parasite plays a key role in development of resistance and thus in treatment failure [9]. The three aquaglyceroporins of T. brucei (TbAQP1, 2, and 3), which are permeable for water and physiological solutes like glycerol and dihydroxyacetone, facilitate the uptake of several non-conventional solutes [10]. For example, TbAQPs are permeable for trivalent antimony (Sb III) and arsenic (As III), which are basic components of drugs used against trypanosomatids [10,11–13]. The latter compound is the chemical basis of melarsoprol. Interestingly, it has been demonstrated that the decrease of TbAQP2 protein causes melarsoprol and pentamidine resistance [14–16]. Although it is
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unknown how TbAQP2 facilitates melarsoprol transport, the mechanism for pentamidine uptake was recently defined. The amidine motif of pentamidine interacts with the highly-conserved asparagine next to the selectivity filter of TbAQP2 blocking the pore and, the drug uptake is due to endocytosis [17]. For almost 100 years boron has been recognized as an essential element in plants, however, only recently, it has been established that boron intake could be beneficial for animals and humans [18,19]. This fact is not particularly surprising as boron is found in many fruits and vegetables and, thus, is regularly consumed by animals and humans (0.35 to 3.0 mg/day) [19]. Interestingly, the boric acid concentration in human blood is at micromolar range [20], therefore, T. brucei is constantly exposed to this compound. Considering all these facts, we decided to investigate using two heterologous expression systems, the permeability of Trypanosoma brucei aquaglyceroporins to boric acid, and we found that TbAQPs are highly permeable to this metalloid in a pH dependent manner.
2. Material and methods 2.1. Ethics Statement This work was performed in accordance with the 'European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes' (Council of Europe No 123, Strasbourg 1985). All experiments were conducted according to German law for the welfare of animals and the surgical techniques on the adult female Xenopus laevis frogs. The procedures were reviewed and approved by the governmental authority of the state Baden-Württemberg (Regierungspräsidium) prior to the start of the study (Anzeige für Organentnahme nach §36).
2.2. Saccharomyces cerevisiae - heterologous expression of TbAQP1, TbAQP2, and TbAQP3 and phenotype experiments 2.2.1. Heterologous expression The yeast strain transformed in this study, BY4742 (MATα; his3Δ 1; leu2Δ 0; lys2Δ 0; ura3Δ 0; YLL043w::kanMX4), which lacks its own aquaglyceroporins, was acquired from Euroscarf (Frankfurt, Germany). All procedures were performed according to standard protocols in yeast genetics [11,21–23]. Briefly, a rich yeast peptone medium containing 2% D-glucose (YPD) was used for the growth of non-transformed yeast. After transformation with empty plasmid (control cells) or constructs (pRS416/TbAQP1, 2 and 3), selection and growth of transformants were done on synthetic medium, namely, complete medium (CM), lacking uracil (the selection marker) and supplemented with 2% glucose as carbon source. 2% agar was used for the solid medium preparation. The pRS416 plasmid possesses a MET25-promoter, and then, the expression of TbAQP 1 and 3 was induced by removing methionine from the culture medium and cells were maintained at room temperature. For TbAQP2 expression, CM medium containing methionine and incubation at 34 °C were used (see results for details).
2.2.2. Phenotype experiments The phenotype was analysed by a plate growth assay. After overnight growth, yeast cultures were adjusted to 1 OD (600 nm) and diluted in a series of 1:10 steps. 10 μl of each dilution step was spotted onto selective agar CM-medium plates without (control condition) or containing additional boric acid (experimental condition). pH of media was adjusted according to the experiment. Yeast cells were grown for 2–4 days at room temperature (for TbAQP1 and 3) or 34 °C (for TbAQP2 see results for details) and their growth was documented by photography.
2.3. Xenopus laevis oocytes - heterologous expression of TbAQP1, TbAQP2, and TbAQP3 and boric acid uptake experiments The oocytes obtained by surgery were defolliculated by treatment with collagenase. Thereafter, stages V–VI oocytes were selected manually and maintained in ND96 solution for 24 h at 16 °C until complementary RNA (cRNA) was injected [11,24,25]. For TbAQPs RNA production by transcription from the specific single stranded template-plasmid, so-called cRNA, Tbaqp 1, 2, and 3 genes subcloned into the pT7T3 expression vector [11], was linearized with the restriction enzyme Sma I. cRNAs were transcribed in vitro and isolated using the mMessage mMachine® kit (Ambion) and RNA clean up kit (Qiagen), respectively. For expression in oocytes, 50 nl of water (control) or 50 nl of water containing 10 ng and 5 ng of cRNA from hAQP1 and one of TbAQPs, respectively, were injected per oocyte. Oocytes were maintained at 16 °C in ND96 buffer containing 5 mM HEPES, pH 7.4, 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate, 0.5 mM theophylline, and 2 μg/ml of gentamicin sulfate. After three days, when the highest expression level of TbAQPs was reached, the standard oocyte swelling assay was performed to determine boric acid permeability [11,25]. The standard iso-osmotic oocyte swelling assay was performed as described by Hansen et al. (2002), Uzcategui et al. (2004), and Figarella et al. (2007) [11,25,26]. TbAQPs were co-expressed in oocytes with the orthodox (specific for water) highly permeable hAQP1. In this way the oocyte swelling assay depends only on the solute permeability i.e., their values will not be limited by water permeability of assayed aquaglyceroporins. Briefly, oocytes were equilibrated for at least 10 min in ND96 (without pyruvate, theophylline, and gentamicin) before the permeability assay was started. Measurements initiated immediately after oocytes were placed in a modified ND96 solution, where 65 mM NaCl was replaced with 130 mM boric acid (this modification in the solution creates a boric acid gradient maintaining the iso-osmotic condition). Swelling assays were carried out at room temperature and were video monitored using a 2.5× objective. The relative volume change within the measurement period was determined from the covered area of the oocyte [11,25,26]. Boric acid permeability (Ps) was calculated using the following equation: Ps ¼ ½osmtotal V0 dðV=V0 Þ=dt=½S ðsolout −solin Þ [27] where osmtotal is the total osmolarity of the system (200 mOsm), V0 is the initial oocyte volume (=9 × 10−4 cm3), S is the oocyte surface area (= 0.045 cm2), solout − solin is the osmotic boric acid gradient and d(V / V0) / dt is measured from the initial slope of the relative volume increase. 3. Results 3.1. Phenotypes of Δfps1 S. cerevisiae strain expressing TbAQPs in presence of boric acid To test initially whether aquaglyceroporins from T. brucei are permeable for boric acid, TbAQP1, 2 and 3 were expressed heterologously in the aquaglyceroporin deficient S. cerevisiae yeast mutant strain (Δfps1). Δfps1 S. cerevisiae strain transformed with plasmid alone served as control. Transformant yeast were induced using a selective medium without methionine (methionine promoter). In order to analyse phenotypes, TbAQPs-transformed Δfps1 yeast and control cells were grown under control conditions (selective medium without boric acid) or challenged with different concentrations of boric acid (Fig. 1). Growth sensitivity to boric acid of Δfps1 yeast cells expressing TbAQPs, but not of control cells, was interpreted as TbAQPs dependent uptake of this compound. As expected, TbAQP1- and 3-transformed Δfps1 yeast and control cells cultivated in control condition, with or without induction, grew
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Fig. 1. Phenotypes of Δfps1 yeast cells transformed with pRS416 alone or pRS416/TbAQP1 or pRS416/TbAQP3 constructs. The induction of expression of TbAQP1 and TbAQP3 was performed using growth medium CM plates without methionine. NaCl and boric acid (BA) concentration were as described in the figure. NaCl was used to control TbAQPs expression.
similarly (Fig. 1, lines 1 and 2). As positive control, to confirm expression and activity of TbAQPs, transformant yeast cells were exposed to hyperosmotic shock with NaCl. To cope with this condition, wild type yeast accumulates glycerol and prevents its efflux by closing its own aquaglyceroporin (FPS1). Δfps1 yeast mutant grows also under hyperosmotic conditions as it can also accumulate glycerol [28]. However, Δfps1 yeast expressing TbAQPs shows a hyper-osmosensitivity phenotype, as these cells are not able to regulate TbAQPs and, therefore, glycerol is constitutively released to the medium [11]. As anticipated, TbAQP1- and 3-transformed Δfps1 yeast cells were strongly affected at 3% NaCl whereas the control cells grew well (Fig. 1, line 3). Interestingly, TbAQP1- and 3-transformants showed a growth reduction in the presence of boric acid, which was dependent on the compound concentration. In contrast, the growth of control cells was not affected by boric acid at any concentration used (Fig. 1, lines 4-8). Therefore, TbAQP1- and 3 are able to transport boric acid. TbAQP2-transformant yeasts exhibited a reduction of the growth in control condition, whenever they were induced to express TbAQP2. When these yeast cells were cultivated in selective medium without methionine, the growth rate was slow (results not shown). This behaviour precludes the evaluation of any phenotype showing that TbAQP2 is considerably toxic for the cells, at least under the chosen experimental conditions. In order to eliminate the adverse effect of TbAQP2 on yeast cell growth, its expression was strongly reduced. To this end, TbAQP2-
transformants were cultivated to suppress the promoter, in presence of methionine, and incubated at 34 °C. Although the TbAQP2 expression was apparently abolished by methionine, significant protein was expressed at this relative high temperature as the plasmid become leaky. In this form, TbAQP2-transformants grew at the same rate as the control cells under control conditions (Fig. 2, line 1). Moreover, they were sensitive to 3% NaCl, whereas control cells were not (Fig. 2, line 2), confirming that TbAQP2 was expressed and active. TbAQP2 transformants exhibited a concentration-dependent sensitivity to boric acid (Fig. 2, lines 3-6). Interestingly, they were more sensitive than TbAQP1- and 3-transformants, subjected to the same conditions, indicating a very high boric acid permeability of TbAQP2 (Fig. 2, lines 3-6). Possible enhancement of the phenotype due to remnant toxicity was ruled out as TbAQP2-transformants were less affected than yeast transformed with TbAQP1 or 3 when they were exposed to 3% NaCl (positive control conditions) (Fig. 2, line 2).
3.2. Uptake of boric acid by Xenopus laevis oocytes expressing TbAQPs In order to confirm the boric acid uptake phenotype in yeast cells and for further characterization, boric acid transport was evaluated in Xenopus laevis oocytes expressing TbAQPs. Boric acid permeability by these cells was determined by an iso-osmotic swelling assay as
Fig. 2. Phenotypes of Δfps1 yeast cells transformed with pRS416 alone or pRS416/TbAQP1, pRS416/TbAQP2, or pRS416/TbAQP3 constructs. The induction of TbAQP1, TbAQP2, and TbAQP3 expression was performed using growth medium CM plates containing methionine and incubated at 34 °C. NaCl and boric acid (BA) concentration were as described in the figure. NaCl was used to control TbAQPs expression.
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boric acid permeability (µm s-1)
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 control
TbAQP1
TbAQP2
TbAQP3
Fig. 3. Boric acid permeability in Xenopus laevis oocytes expressing TbAQP1-3. Oocytes were injected with water (control) or with a combination of 5 ng of hAQP1-cRNA plus 10 ng of TbAQP1, 2 or 3 – cRNA. After three days of incubation, permeability experiments were performed. For each condition (control, hAQP1/TbAQP1, hAQP1/ TbAQP2, and hAQP1/TbAQP3) between 6–8 oocytes per batch were measured and at least 3 oocyte batches were evaluated. The results were represented as mean ± SE.
described in material and methods. Oocytes injected with water (control cells) were used to determine any basal uptake. As shown in Fig. 3, control cells displayed negligible swelling when they were challenged with an inwardly directed boric acid gradient. The permeability values calculated were very low (Ps = 0.079 μm/s) consistent with simple membrane diffusion. By contrast, oocytes injected with TbAQP1-, 2- or 3-cRNA displayed a high swelling rate in the following order TbAQP2 N TbAQP3 N TbAQ1. Expression of TbAQP2 in Xenopus laevis oocytes generated a 15.5-fold increase of the swelling rate, as compared with water-injected oocytes, which represents a permeability value of 1.221 μm/s. In the presence of the boric acid gradient, TbAQP3 showed more boric acid passage through oocytes membrane than that observed in water-injected oocytes, reflecting a 6.7-fold (Ps = 0.5 μm/s) oocyte permeability compared with the control. Under the same conditions, TbAQP1 permeability was lower than that of TbAQP2 and TbAQP3, but still increased boric acid permeability 2.6fold as compared with oocyte controls (Ps = 0.208 μm/s). These results, along with those in yeast, demonstrated undoubtedly that T. brucei aquaglyceroporins are boric acid channels.
Fig. 4. Boric acid pH-dependent uptake in Δfps1 mutant cells expressing TbAQP1 and 3. Yeast cell mutants expressing or not-expressing TbAQP1 and 3 were spotted onto selectiveinduction agar medium under different conditions. Media containing 9 or 11 mM boric acid was prepared at different pH from 3.5 to 6.5. Growth control condition does not have boric acid. Positive control, for channel activity evaluation, was performed adding 1% and 3% of NaCl into the medium. The induction of protein expression was performed using growth medium CM plates without methionine
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3.3. Effect of pH on the TbAQPs permeability for boric acid Boron is a mineral abundantly present in nature, and exists in two forms, as charged borate (B(OH)− 4 ) and as uncharged boric acid (H3BO3). With a pKa of 9.25 boric acid is the dominant structure for boron in a pH range from acidic to moderately alkaline [20]. We decided to evaluate the pH-dependence of TbAQPs permeability for boric acid, as any change in the channel activity should reflect mainly the fine changes in the protein structure. Although TbAQPs are very similar to each other (77% identity) [11], they exhibit important differences in the pore structure [11,29]. TbAQP1 and TbAQP3 possess two canonical NPA motifs and the same amino acids (WGYR) in the aromatic/arginine (ar/R) constriction, residues mainly responsible for solutes permeability. TbAQP2 contains unusual NSA/NPS motifs, does not possess the highly conserved arginine at its (ar/R) region, and all associated amino acids are hydrophobic (IVLL). Therefore, pH sensitivity was expected to differ between TbAQP1 and TbAQP3 with respect to TbAQP2. As illustrated in Fig. 4, yeast cells expressing TbAQP1 and 3 showed sensitivity to changes in pH. At the highest pH tested (pH 6.5) these cells displayed a strong growth reduction, and when the pH was decreased progressively to pH 4.5 they steadily increased their growth. Interestingly, at pH 3.5, a prominent decrease in growth was noted comparable with that occurred at pH 6.5. Any pH unspecific effect on the yeasts growth was ruled out as the controls cells grew well in this condition (Fig. 4). Even though the boric acid permeability of TbAQP2-transformed Δfps1 mutant cells was also affected by pH, the pattern was slightly different from that of TbAQP1 and 3 transformants (Fig. 5). Δfps1/TbAQP2 mutants showed a strong defect in their growth at pH 6.5, which was disappeared upon decrease of pH to the lowest pH evaluated (pH 3.5). These results may be due to structural differences in amino acid composition of the channels. There are three identical regions between TbAQP1 and 3 that differ from TbAQP2, which are relevant for the
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selective permeability and could be responsible for the differential effect. These regions are NSA/NPS motifs, the ar/R constriction, and some amino acids in the loop C, which could be affected by the pH, as they are whether part of the pore or exposed to the extracellular space. 4. Discussion African Trypanosomes have three aquaglyceroporins localised differentially in the parasite, but all of them are in contact with the environment [15,30]. They have been mainly characterized as water/glycerol/dihydroxyacetone channels, with limited capacity of transporting polyols [11]. It was also shown that these channels have a wide permeability profile for small non-charged solutes. Ammonia and antimonite as well as arsenite (basic components of numerous drugs against trypanosomatids) pass also through TbAQPs [10,12]. Recently, several lines of evidence showed that TbAQP2 facilitate the entrance, in some way, into the parasite of the drugs commonly used to treat sleeping sickness: pentamidine and melarsoprol [14,16,17,31]. Boric acid permeability in aquaglyceroporins has been mostly documented in plants [32]. In other organisms the information is scarce, only a few examples are known. In yeast, FPS1 is permeable for boric acid and this element is required for normal proliferation [33]. Interestingly, T. brucei lives in blood containing boric acid at μM concentrations [20,34]. Therefore, it may be by itself beneficial for the parasite, as it has been reported for humans, plants, yeast, and other organisms [20]. However, it is completely unknown if T. brucei uses boron for its metabolism. The discovery of an entry pathway for boric acid is the first step to shed light on the possible significance of the boron element for the parasite. We have demonstrated, by using two different heterologous expression systems, that TbAQPs are permeable for boric acid. TbAQP2 showed the highest permeability, which was comparable to the highest transport rate of solutes known for TbAQPs [11,29]. TbAQP2 boric acid
Fig. 5. Boric acid pH-dependent permeability in Δfps1 mutant cells expressing TbAQP2. Yeast cell mutants expressing TbAQP2 and control cells were spotted onto selective-induction agar medium under different conditions. Media containing 9 or 11 mM boric acid was prepared at different pH from 3.5 to 6.5. Growth control condition does not have boric acid. Positive control, for channel activity evaluation, was performed adding 1% and 3% of NaCl into the medium. The induction of TbAQP2 expression was performed using growth medium CM plates containing methionine and incubated at 34 °C.
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permeability was twice as high as TbAQP3 and almost 6-fold higher than TbAQP1. In principle, the factor defining the channel permeability is the amino acid composition of the ar/R constriction [35–39]. In plant aquaglyceroporins this region is directly related to boric acid passage [40]. A wider and hydrophobic ar/R constriction region tends to facilitate solute permeation; in TbAQP2, it seems to be the case, as some bulky aromatic amino acids in this region are missing, which probably causes a wider constriction region [9,29]. This region is also highly hydrophobic, due to the absence of arginine and presence of IVLL amino acids. Altogether, these features in the selectivity filter of TbAQP2 may create an enabling environment, for more effective boric acid transport. Interestingly, TbAQP1 and 3 showed appreciable differences regarding the boric acid permeability. These aquaglyceroporins have the same ar/R constriction region, as well as highly conserved NPA motifs, the second most important constriction area in the pore. Therefore, these regions are not the only ones responsible for the selectivity of this compound, and a more complex pattern of organisation in the protein should determine the final conductance of boric acid. It has been established that other regions besides the ar/R filter and NPA motif can help in improving the fine-tuning of solute permeability. When the amino acid composition is modified, in extra- or intracellular connecting loops of aquaglyceroporins from protozoa as Dictyostelium, Plasmodium, and Leishmania, the permeability for water, glycerol or metalloids change differentially [41–44]. TbAQPs are 77% identical in their amino acid composition, however, TbAQP2 and 3 are phylogenetically more related [11]. They share 75% identity whereas TbAQP1 and 2 share 56%. The key difference between TbAQP1 and TbAQP2 and 3, is that the former has a completely different long N-terminus. This feature could be associated with different functions, one of which could be the regulation of the permeability for solutes like boric acid. In human AQP5 and spinach (Spinacia oleracea) leaf aquaporins, the N-terminal participates in the strict regulation of the water passage [39]. Moreover, N-terminus phosphorylation in a plant aquaporin (NbXIP1;1 from Nicotiana benthamiana) has recently described as regulator of boric acid permeability [45]. We also showed that boric acid TbAQPs permeability was affected by changes in pH. A slightly different behaviour between TbAQP1 and 3 with respect to TbAQP2 was also observed. pH dependence of arsenite and antimonite transport by aquaglyceroporins from T. brucei has been reported [10]. As for arsenite and antimonite, low pH decreases the permeability of TbAQPs for boric acid. Taken together, T. brucei aquaglyceroporins are a very efficient entry pathway for boric acid into the parasite. Particularly TbAQP2 is similarly permeable for this compound as for dihydroxyacetone and glycerol. This property could be used in the future to improve the design of boron-containing drugs, as boric acid could work as a high-affinity ligand for these aquaglyceroporins and then facilitate the uptake of more complex drugs by endocytosis. To our knowledge this is the first time that aquaglyceroporins from protozoa have been identified as boron-channels and we expect that more aquaglyceroporins from these single-cells will be discovered as having this transport capacity, particularly, due to the potential of this compound as a nutritional element. Therefore, our findings pave the road not only to understand better the physiological and pharmacological importance of boron in T. brucei, but also for other protozoa. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This work was supported by Consejo de Desarrollo Científico y Humanístico, Universidad Central de Venezuela (PG 09-7059-2007/2), and FONACIT, Venezuela (No. 2013001630). NLU received a fellowship
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Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei Sabrina Marsiccobetre a, Alexis Rodríguez-Acosta a, Florian Lang b, Katherine Figarella a,⁎, Néstor L. Uzcátegui a,b,⁎ a b
Laboratory of Immunochemistry and Ultrastructure, Institute of Anatomy, Central University of Venezuela, Caracas, Venezuela Department of Physiology I, University of Tubingen, Tubingen, Germany
a r t i c l e
i n f o
Article history: Received 5 October 2016 Received in revised form 3 January 2017 Accepted 7 January 2017 Available online 10 January 2017 Keywords: aquaglyceroporins boric acid Trypanosoma brucei heterologous expression
a b s t r a c t The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Diseases caused by members of the Trypanosomatidae family depict a relevant source of mortality and morbidity worldwide and especially in developing countries [1–3]. One of these diseases is the human African trypanosomiasis (HAT), or sleeping sickness, caused by two sub species of Trypanosoma brucei (T. b. gambiense and T. b. rhodesiense), which threatens millions of people in 36 countries in the sub-Saharan region and has a mortality rate nearly 100%, if it is not treated [4]. There are few drugs for the treatment of HAT, and unfortunately, most of them are highly toxic. Pentamidine and Suramin produce severe side effects and are ineffective in the chronic phase. The EflornithineNifurtimox combination is an acceptable treatment for the chronic stage of HAT caused by T. b. gambiense [5]. Meanwhile, Melarsoprol (Arsenal®, Aventis), a highly toxic arsenic derivative, is the only effective therapy against the chronic stage in patients infected with T. b. rhodesiense. Nevertheless, near 10% of patients treated with this drug develop a severe encephalopathy, which is fatal in half of the ⁎ Corresponding authors at: Universidad Central de Venezuela. Paseo Los Ilustres, Ciudad Universitaria. Instituto Anatómico. Caracas 1040, Venezuela. E-mail addresses: kfi[email protected] (K. Figarella), [email protected] (N.L. Uzcátegui).
http://dx.doi.org/10.1016/j.bbamem.2017.01.011 0005-2736/© 2017 Elsevier B.V. All rights reserved.
cases. Moreover, treatment failure of these drugs due to resistance is common [6]. Development of new more effective and safe trypanocidal compounds is urgently needed. Boron-containing molecules seem to be an interesting option, as they are effective against a wide range of infectious diseases [7]. The drugs for Neglected Diseases initiative (DNDi) organization, Anacor Pharmaceuticals, and their partners have developed several promising boron-containing molecules, the so-called oxaboroles. Several of them are highly active against HAT chronic phase in animal models. One of the oxaboroles, SCYX-7158, has already completed phase I clinical trials (DNDi). However, the action mechanisms of boron containing molecules are unknown [8]. Down regulation of membrane proteins responsible for drug transport into the parasite plays a key role in development of resistance and thus in treatment failure [9]. The three aquaglyceroporins of T. brucei (TbAQP1, 2, and 3), which are permeable for water and physiological solutes like glycerol and dihydroxyacetone, facilitate the uptake of several non-conventional solutes [10]. For example, TbAQPs are permeable for trivalent antimony (Sb III) and arsenic (As III), which are basic components of drugs used against trypanosomatids [10,11–13]. The latter compound is the chemical basis of melarsoprol. Interestingly, it has been demonstrated that the decrease of TbAQP2 protein causes melarsoprol and pentamidine resistance [14–16]. Although it is
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unknown how TbAQP2 facilitates melarsoprol transport, the mechanism for pentamidine uptake was recently defined. The amidine motif of pentamidine interacts with the highly-conserved asparagine next to the selectivity filter of TbAQP2 blocking the pore and, the drug uptake is due to endocytosis [17]. For almost 100 years boron has been recognized as an essential element in plants, however, only recently, it has been established that boron intake could be beneficial for animals and humans [18,19]. This fact is not particularly surprising as boron is found in many fruits and vegetables and, thus, is regularly consumed by animals and humans (0.35 to 3.0 mg/day) [19]. Interestingly, the boric acid concentration in human blood is at micromolar range [20], therefore, T. brucei is constantly exposed to this compound. Considering all these facts, we decided to investigate using two heterologous expression systems, the permeability of Trypanosoma brucei aquaglyceroporins to boric acid, and we found that TbAQPs are highly permeable to this metalloid in a pH dependent manner.
2. Material and methods 2.1. Ethics Statement This work was performed in accordance with the 'European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes' (Council of Europe No 123, Strasbourg 1985). All experiments were conducted according to German law for the welfare of animals and the surgical techniques on the adult female Xenopus laevis frogs. The procedures were reviewed and approved by the governmental authority of the state Baden-Württemberg (Regierungspräsidium) prior to the start of the study (Anzeige für Organentnahme nach §36).
2.2. Saccharomyces cerevisiae - heterologous expression of TbAQP1, TbAQP2, and TbAQP3 and phenotype experiments 2.2.1. Heterologous expression The yeast strain transformed in this study, BY4742 (MATα; his3Δ 1; leu2Δ 0; lys2Δ 0; ura3Δ 0; YLL043w::kanMX4), which lacks its own aquaglyceroporins, was acquired from Euroscarf (Frankfurt, Germany). All procedures were performed according to standard protocols in yeast genetics [11,21–23]. Briefly, a rich yeast peptone medium containing 2% D-glucose (YPD) was used for the growth of non-transformed yeast. After transformation with empty plasmid (control cells) or constructs (pRS416/TbAQP1, 2 and 3), selection and growth of transformants were done on synthetic medium, namely, complete medium (CM), lacking uracil (the selection marker) and supplemented with 2% glucose as carbon source. 2% agar was used for the solid medium preparation. The pRS416 plasmid possesses a MET25-promoter, and then, the expression of TbAQP 1 and 3 was induced by removing methionine from the culture medium and cells were maintained at room temperature. For TbAQP2 expression, CM medium containing methionine and incubation at 34 °C were used (see results for details).
2.2.2. Phenotype experiments The phenotype was analysed by a plate growth assay. After overnight growth, yeast cultures were adjusted to 1 OD (600 nm) and diluted in a series of 1:10 steps. 10 μl of each dilution step was spotted onto selective agar CM-medium plates without (control condition) or containing additional boric acid (experimental condition). pH of media was adjusted according to the experiment. Yeast cells were grown for 2–4 days at room temperature (for TbAQP1 and 3) or 34 °C (for TbAQP2 see results for details) and their growth was documented by photography.
2.3. Xenopus laevis oocytes - heterologous expression of TbAQP1, TbAQP2, and TbAQP3 and boric acid uptake experiments The oocytes obtained by surgery were defolliculated by treatment with collagenase. Thereafter, stages V–VI oocytes were selected manually and maintained in ND96 solution for 24 h at 16 °C until complementary RNA (cRNA) was injected [11,24,25]. For TbAQPs RNA production by transcription from the specific single stranded template-plasmid, so-called cRNA, Tbaqp 1, 2, and 3 genes subcloned into the pT7T3 expression vector [11], was linearized with the restriction enzyme Sma I. cRNAs were transcribed in vitro and isolated using the mMessage mMachine® kit (Ambion) and RNA clean up kit (Qiagen), respectively. For expression in oocytes, 50 nl of water (control) or 50 nl of water containing 10 ng and 5 ng of cRNA from hAQP1 and one of TbAQPs, respectively, were injected per oocyte. Oocytes were maintained at 16 °C in ND96 buffer containing 5 mM HEPES, pH 7.4, 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate, 0.5 mM theophylline, and 2 μg/ml of gentamicin sulfate. After three days, when the highest expression level of TbAQPs was reached, the standard oocyte swelling assay was performed to determine boric acid permeability [11,25]. The standard iso-osmotic oocyte swelling assay was performed as described by Hansen et al. (2002), Uzcategui et al. (2004), and Figarella et al. (2007) [11,25,26]. TbAQPs were co-expressed in oocytes with the orthodox (specific for water) highly permeable hAQP1. In this way the oocyte swelling assay depends only on the solute permeability i.e., their values will not be limited by water permeability of assayed aquaglyceroporins. Briefly, oocytes were equilibrated for at least 10 min in ND96 (without pyruvate, theophylline, and gentamicin) before the permeability assay was started. Measurements initiated immediately after oocytes were placed in a modified ND96 solution, where 65 mM NaCl was replaced with 130 mM boric acid (this modification in the solution creates a boric acid gradient maintaining the iso-osmotic condition). Swelling assays were carried out at room temperature and were video monitored using a 2.5× objective. The relative volume change within the measurement period was determined from the covered area of the oocyte [11,25,26]. Boric acid permeability (Ps) was calculated using the following equation: Ps ¼ ½osmtotal V0 dðV=V0 Þ=dt=½S ðsolout −solin Þ [27] where osmtotal is the total osmolarity of the system (200 mOsm), V0 is the initial oocyte volume (=9 × 10−4 cm3), S is the oocyte surface area (= 0.045 cm2), solout − solin is the osmotic boric acid gradient and d(V / V0) / dt is measured from the initial slope of the relative volume increase. 3. Results 3.1. Phenotypes of Δfps1 S. cerevisiae strain expressing TbAQPs in presence of boric acid To test initially whether aquaglyceroporins from T. brucei are permeable for boric acid, TbAQP1, 2 and 3 were expressed heterologously in the aquaglyceroporin deficient S. cerevisiae yeast mutant strain (Δfps1). Δfps1 S. cerevisiae strain transformed with plasmid alone served as control. Transformant yeast were induced using a selective medium without methionine (methionine promoter). In order to analyse phenotypes, TbAQPs-transformed Δfps1 yeast and control cells were grown under control conditions (selective medium without boric acid) or challenged with different concentrations of boric acid (Fig. 1). Growth sensitivity to boric acid of Δfps1 yeast cells expressing TbAQPs, but not of control cells, was interpreted as TbAQPs dependent uptake of this compound. As expected, TbAQP1- and 3-transformed Δfps1 yeast and control cells cultivated in control condition, with or without induction, grew
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Fig. 1. Phenotypes of Δfps1 yeast cells transformed with pRS416 alone or pRS416/TbAQP1 or pRS416/TbAQP3 constructs. The induction of expression of TbAQP1 and TbAQP3 was performed using growth medium CM plates without methionine. NaCl and boric acid (BA) concentration were as described in the figure. NaCl was used to control TbAQPs expression.
similarly (Fig. 1, lines 1 and 2). As positive control, to confirm expression and activity of TbAQPs, transformant yeast cells were exposed to hyperosmotic shock with NaCl. To cope with this condition, wild type yeast accumulates glycerol and prevents its efflux by closing its own aquaglyceroporin (FPS1). Δfps1 yeast mutant grows also under hyperosmotic conditions as it can also accumulate glycerol [28]. However, Δfps1 yeast expressing TbAQPs shows a hyper-osmosensitivity phenotype, as these cells are not able to regulate TbAQPs and, therefore, glycerol is constitutively released to the medium [11]. As anticipated, TbAQP1- and 3-transformed Δfps1 yeast cells were strongly affected at 3% NaCl whereas the control cells grew well (Fig. 1, line 3). Interestingly, TbAQP1- and 3-transformants showed a growth reduction in the presence of boric acid, which was dependent on the compound concentration. In contrast, the growth of control cells was not affected by boric acid at any concentration used (Fig. 1, lines 4-8). Therefore, TbAQP1- and 3 are able to transport boric acid. TbAQP2-transformant yeasts exhibited a reduction of the growth in control condition, whenever they were induced to express TbAQP2. When these yeast cells were cultivated in selective medium without methionine, the growth rate was slow (results not shown). This behaviour precludes the evaluation of any phenotype showing that TbAQP2 is considerably toxic for the cells, at least under the chosen experimental conditions. In order to eliminate the adverse effect of TbAQP2 on yeast cell growth, its expression was strongly reduced. To this end, TbAQP2-
transformants were cultivated to suppress the promoter, in presence of methionine, and incubated at 34 °C. Although the TbAQP2 expression was apparently abolished by methionine, significant protein was expressed at this relative high temperature as the plasmid become leaky. In this form, TbAQP2-transformants grew at the same rate as the control cells under control conditions (Fig. 2, line 1). Moreover, they were sensitive to 3% NaCl, whereas control cells were not (Fig. 2, line 2), confirming that TbAQP2 was expressed and active. TbAQP2 transformants exhibited a concentration-dependent sensitivity to boric acid (Fig. 2, lines 3-6). Interestingly, they were more sensitive than TbAQP1- and 3-transformants, subjected to the same conditions, indicating a very high boric acid permeability of TbAQP2 (Fig. 2, lines 3-6). Possible enhancement of the phenotype due to remnant toxicity was ruled out as TbAQP2-transformants were less affected than yeast transformed with TbAQP1 or 3 when they were exposed to 3% NaCl (positive control conditions) (Fig. 2, line 2).
3.2. Uptake of boric acid by Xenopus laevis oocytes expressing TbAQPs In order to confirm the boric acid uptake phenotype in yeast cells and for further characterization, boric acid transport was evaluated in Xenopus laevis oocytes expressing TbAQPs. Boric acid permeability by these cells was determined by an iso-osmotic swelling assay as
Fig. 2. Phenotypes of Δfps1 yeast cells transformed with pRS416 alone or pRS416/TbAQP1, pRS416/TbAQP2, or pRS416/TbAQP3 constructs. The induction of TbAQP1, TbAQP2, and TbAQP3 expression was performed using growth medium CM plates containing methionine and incubated at 34 °C. NaCl and boric acid (BA) concentration were as described in the figure. NaCl was used to control TbAQPs expression.
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boric acid permeability (µm s-1)
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 control
TbAQP1
TbAQP2
TbAQP3
Fig. 3. Boric acid permeability in Xenopus laevis oocytes expressing TbAQP1-3. Oocytes were injected with water (control) or with a combination of 5 ng of hAQP1-cRNA plus 10 ng of TbAQP1, 2 or 3 – cRNA. After three days of incubation, permeability experiments were performed. For each condition (control, hAQP1/TbAQP1, hAQP1/ TbAQP2, and hAQP1/TbAQP3) between 6–8 oocytes per batch were measured and at least 3 oocyte batches were evaluated. The results were represented as mean ± SE.
described in material and methods. Oocytes injected with water (control cells) were used to determine any basal uptake. As shown in Fig. 3, control cells displayed negligible swelling when they were challenged with an inwardly directed boric acid gradient. The permeability values calculated were very low (Ps = 0.079 μm/s) consistent with simple membrane diffusion. By contrast, oocytes injected with TbAQP1-, 2- or 3-cRNA displayed a high swelling rate in the following order TbAQP2 N TbAQP3 N TbAQ1. Expression of TbAQP2 in Xenopus laevis oocytes generated a 15.5-fold increase of the swelling rate, as compared with water-injected oocytes, which represents a permeability value of 1.221 μm/s. In the presence of the boric acid gradient, TbAQP3 showed more boric acid passage through oocytes membrane than that observed in water-injected oocytes, reflecting a 6.7-fold (Ps = 0.5 μm/s) oocyte permeability compared with the control. Under the same conditions, TbAQP1 permeability was lower than that of TbAQP2 and TbAQP3, but still increased boric acid permeability 2.6fold as compared with oocyte controls (Ps = 0.208 μm/s). These results, along with those in yeast, demonstrated undoubtedly that T. brucei aquaglyceroporins are boric acid channels.
Fig. 4. Boric acid pH-dependent uptake in Δfps1 mutant cells expressing TbAQP1 and 3. Yeast cell mutants expressing or not-expressing TbAQP1 and 3 were spotted onto selectiveinduction agar medium under different conditions. Media containing 9 or 11 mM boric acid was prepared at different pH from 3.5 to 6.5. Growth control condition does not have boric acid. Positive control, for channel activity evaluation, was performed adding 1% and 3% of NaCl into the medium. The induction of protein expression was performed using growth medium CM plates without methionine
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3.3. Effect of pH on the TbAQPs permeability for boric acid Boron is a mineral abundantly present in nature, and exists in two forms, as charged borate (B(OH)− 4 ) and as uncharged boric acid (H3BO3). With a pKa of 9.25 boric acid is the dominant structure for boron in a pH range from acidic to moderately alkaline [20]. We decided to evaluate the pH-dependence of TbAQPs permeability for boric acid, as any change in the channel activity should reflect mainly the fine changes in the protein structure. Although TbAQPs are very similar to each other (77% identity) [11], they exhibit important differences in the pore structure [11,29]. TbAQP1 and TbAQP3 possess two canonical NPA motifs and the same amino acids (WGYR) in the aromatic/arginine (ar/R) constriction, residues mainly responsible for solutes permeability. TbAQP2 contains unusual NSA/NPS motifs, does not possess the highly conserved arginine at its (ar/R) region, and all associated amino acids are hydrophobic (IVLL). Therefore, pH sensitivity was expected to differ between TbAQP1 and TbAQP3 with respect to TbAQP2. As illustrated in Fig. 4, yeast cells expressing TbAQP1 and 3 showed sensitivity to changes in pH. At the highest pH tested (pH 6.5) these cells displayed a strong growth reduction, and when the pH was decreased progressively to pH 4.5 they steadily increased their growth. Interestingly, at pH 3.5, a prominent decrease in growth was noted comparable with that occurred at pH 6.5. Any pH unspecific effect on the yeasts growth was ruled out as the controls cells grew well in this condition (Fig. 4). Even though the boric acid permeability of TbAQP2-transformed Δfps1 mutant cells was also affected by pH, the pattern was slightly different from that of TbAQP1 and 3 transformants (Fig. 5). Δfps1/TbAQP2 mutants showed a strong defect in their growth at pH 6.5, which was disappeared upon decrease of pH to the lowest pH evaluated (pH 3.5). These results may be due to structural differences in amino acid composition of the channels. There are three identical regions between TbAQP1 and 3 that differ from TbAQP2, which are relevant for the
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selective permeability and could be responsible for the differential effect. These regions are NSA/NPS motifs, the ar/R constriction, and some amino acids in the loop C, which could be affected by the pH, as they are whether part of the pore or exposed to the extracellular space. 4. Discussion African Trypanosomes have three aquaglyceroporins localised differentially in the parasite, but all of them are in contact with the environment [15,30]. They have been mainly characterized as water/glycerol/dihydroxyacetone channels, with limited capacity of transporting polyols [11]. It was also shown that these channels have a wide permeability profile for small non-charged solutes. Ammonia and antimonite as well as arsenite (basic components of numerous drugs against trypanosomatids) pass also through TbAQPs [10,12]. Recently, several lines of evidence showed that TbAQP2 facilitate the entrance, in some way, into the parasite of the drugs commonly used to treat sleeping sickness: pentamidine and melarsoprol [14,16,17,31]. Boric acid permeability in aquaglyceroporins has been mostly documented in plants [32]. In other organisms the information is scarce, only a few examples are known. In yeast, FPS1 is permeable for boric acid and this element is required for normal proliferation [33]. Interestingly, T. brucei lives in blood containing boric acid at μM concentrations [20,34]. Therefore, it may be by itself beneficial for the parasite, as it has been reported for humans, plants, yeast, and other organisms [20]. However, it is completely unknown if T. brucei uses boron for its metabolism. The discovery of an entry pathway for boric acid is the first step to shed light on the possible significance of the boron element for the parasite. We have demonstrated, by using two different heterologous expression systems, that TbAQPs are permeable for boric acid. TbAQP2 showed the highest permeability, which was comparable to the highest transport rate of solutes known for TbAQPs [11,29]. TbAQP2 boric acid
Fig. 5. Boric acid pH-dependent permeability in Δfps1 mutant cells expressing TbAQP2. Yeast cell mutants expressing TbAQP2 and control cells were spotted onto selective-induction agar medium under different conditions. Media containing 9 or 11 mM boric acid was prepared at different pH from 3.5 to 6.5. Growth control condition does not have boric acid. Positive control, for channel activity evaluation, was performed adding 1% and 3% of NaCl into the medium. The induction of TbAQP2 expression was performed using growth medium CM plates containing methionine and incubated at 34 °C.
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permeability was twice as high as TbAQP3 and almost 6-fold higher than TbAQP1. In principle, the factor defining the channel permeability is the amino acid composition of the ar/R constriction [35–39]. In plant aquaglyceroporins this region is directly related to boric acid passage [40]. A wider and hydrophobic ar/R constriction region tends to facilitate solute permeation; in TbAQP2, it seems to be the case, as some bulky aromatic amino acids in this region are missing, which probably causes a wider constriction region [9,29]. This region is also highly hydrophobic, due to the absence of arginine and presence of IVLL amino acids. Altogether, these features in the selectivity filter of TbAQP2 may create an enabling environment, for more effective boric acid transport. Interestingly, TbAQP1 and 3 showed appreciable differences regarding the boric acid permeability. These aquaglyceroporins have the same ar/R constriction region, as well as highly conserved NPA motifs, the second most important constriction area in the pore. Therefore, these regions are not the only ones responsible for the selectivity of this compound, and a more complex pattern of organisation in the protein should determine the final conductance of boric acid. It has been established that other regions besides the ar/R filter and NPA motif can help in improving the fine-tuning of solute permeability. When the amino acid composition is modified, in extra- or intracellular connecting loops of aquaglyceroporins from protozoa as Dictyostelium, Plasmodium, and Leishmania, the permeability for water, glycerol or metalloids change differentially [41–44]. TbAQPs are 77% identical in their amino acid composition, however, TbAQP2 and 3 are phylogenetically more related [11]. They share 75% identity whereas TbAQP1 and 2 share 56%. The key difference between TbAQP1 and TbAQP2 and 3, is that the former has a completely different long N-terminus. This feature could be associated with different functions, one of which could be the regulation of the permeability for solutes like boric acid. In human AQP5 and spinach (Spinacia oleracea) leaf aquaporins, the N-terminal participates in the strict regulation of the water passage [39]. Moreover, N-terminus phosphorylation in a plant aquaporin (NbXIP1;1 from Nicotiana benthamiana) has recently described as regulator of boric acid permeability [45]. We also showed that boric acid TbAQPs permeability was affected by changes in pH. A slightly different behaviour between TbAQP1 and 3 with respect to TbAQP2 was also observed. pH dependence of arsenite and antimonite transport by aquaglyceroporins from T. brucei has been reported [10]. As for arsenite and antimonite, low pH decreases the permeability of TbAQPs for boric acid. Taken together, T. brucei aquaglyceroporins are a very efficient entry pathway for boric acid into the parasite. Particularly TbAQP2 is similarly permeable for this compound as for dihydroxyacetone and glycerol. This property could be used in the future to improve the design of boron-containing drugs, as boric acid could work as a high-affinity ligand for these aquaglyceroporins and then facilitate the uptake of more complex drugs by endocytosis. To our knowledge this is the first time that aquaglyceroporins from protozoa have been identified as boron-channels and we expect that more aquaglyceroporins from these single-cells will be discovered as having this transport capacity, particularly, due to the potential of this compound as a nutritional element. Therefore, our findings pave the road not only to understand better the physiological and pharmacological importance of boron in T. brucei, but also for other protozoa. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This work was supported by Consejo de Desarrollo Científico y Humanístico, Universidad Central de Venezuela (PG 09-7059-2007/2), and FONACIT, Venezuela (No. 2013001630). NLU received a fellowship
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