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Crithidia luciliae: Functional Expression of Nucleoside and Nucleobase Transporters inXenopus laevisOocytes
EXPERIMENTAL PARASITOLOGY ARTICLE NO.
90, 181–188 (1998)
PR984320
Crithidia luciliae: Functional Expression of Nucleoside and Nucleobase Transporters in Xenopus laevis Oocytes
Simone T. Hall,*,1 Jeffrey I. Penny,† Annette M. Gero,* and Sanjeev Krishna† *School of Biochemistry and Molecular Genetics, The University of NSW, Sydney, NSW, 2052, Australia; and †Division of Infectious Diseases, St. George’s Hospital Medical School, London, SW17 ORE, U.K.
Hall, S. T., Penny, J. I., Gero, A. M., and Krishna, S. 1998. Crithidia luciliae: Functional expression of nucleoside and nucleobase transporters in Xenopus laevis oocytes. Experimental Parasitology 90, 181–188. The expression of purine-specific nucleoside and base transporters of Crithidia luciliae has been demonstrated in Xenopus laevis oocytes. Poly(A)+-mRNA from C. luciliae, cultured in either purine-replete or purine-starved conditions, was microinjected into X. laevis oocytes. For “purine-replete” mRNA, expression of adenosine and hypoxanthine uptake in microinjected X. laevis oocytes was increased on average 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with “purine-starved” mRNA was 8 and 3-fold above water-injected controls, respectively. Substrate competition indicated an adenosine/deoxyadenosine transporter and a separate base transporter specific for hypoxanthine. In contrast to C. luciliae in vivo, where the level of activity of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters (from both purine replete and deplete cultures) in X. laevis oocytes was independent of the extracellular purine concentration. These results may suggest that the presence of specific transporter message is independent of the extracellular purine content, indicating that the regulation of activation and expression of these transporters in C. luciliae may not be under transcriptional control. q 1998 Academic Press Index Descriptors and Abbreviations: Crithidia luciliae; Xenopus laevis; functional expression; adenosine transporter; hypoxanthine transporter; 38-nucleotidase/nuclease, 38-NTase (EC 3.1.3.6); DEPC, diethylpyrocarbonate; MBM, modified Barths’ medium; 28DOG, 28-deoxy-D-glucose; IQR, interquartile range.
1 To whom correspondence should be addressed. Fax: (612) 93851483. E-mail: [email protected].
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
INTRODUCTION
Parasitic protozoa lack a de novo purine biosynthetic pathway and hence depend on the salvage of purines for nucleic acid synthesis by specific transport proteins. The trypanosomatid Crithidia luciliae is closely related to the pathogenic trypanosomatids Leishmania spp. and Trypanosoma spp. Biochemical evidence indicates that C. luciliae possess at least three distinct purine transport systems. Two of these mediate uptake of purine nucleosides: one for adenosine and its analogues and one for guanosine, its analogues, and inosine (Hall et al. 1993). The third represents a separate base transporter with high affinity for hypoxanthine and adenine; although adenosine is also transported by this system (Day and Gero 1997). Purine transporters with similar substrate specificities have been characterised in L. braziliensis (Hansen et al. 1982), L. donovani promastigotes (Aronow et al. 1987), L. major promastigotes (Baer et al. 1992), T. brucei brucei procyclic and bloodstream forms (deKoning and Jarvis 1997), and C. fasciculata (Kidder et al. 1978). C. luciliae exhibits an unusual phenomenon in which the level of activity of transporters which mediate uptake of purine nucleosides and nucleobases is dependent upon the concentration of available purines in the culture medium. It has been observed that a dramatic increase in the level of adenosine, guanosine, and hypoxanthine transport occurs in C. luciliae grown in purine-deficient medium compared to cells grown in purine-replete medium (Alleman and Gottlieb 1996; Hall et al. 1996; Day and Gero 1997; Gero et al.
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182 1997). The increase in purine uptake observed under purinestarved conditions is reversed by the addition of extracellular purines to the medium (Hall et al. 1996; Day and Gero 1997). This nutrient-induced upregulation of nucleoside/ base transporters is a phenomenon not reported in any other protozoan parasite studied previously. The biological importance of these transporters is evident as protozoan parasites rely on purine salvage pathways to obtain essential nutrients from the extracellular milieu. Only recently the genes encoding two nucleoside transporters of the related parasite L. donovani have been isolated and sequenced (Carter et al. 1997; Vasudevan et al. 1997). We report here the functional expression in Xenopus oocytes of an adenosine and a hypoxanthine transporter from C. luciliae mRNA isolated from either purine-replete or purine-starved cells. These findings represent the first step in the characterisation of the genes encoding purine nucleoside and base transporters of C. luciliae.
MATERIALS AND METHODS
Cell culture. C. luciliae were grown aerobically in RPMI 1640 medium containing 25 mM NaHCO3 (pH 7.4) and supplemented with 2 mM L-glutamine and 10% (v/v) heat-inactivated foetal bovine serum (Trace Biosciences). Cultures were inoculated by the addition of 30 ml stationary phase cells ('2 3 107 cells/ml) to 300 ml complete medium, in 1-L glass culture flasks and maintained at 268C for 24 h for purine-replete cells or 72 h for purine-starved cells as previously described (Hall et al., 1996). Radiochemicals. [8-14C]D-Adenosine (1.85 [U-14C[GBq/mmol), [U-14C]hypoxanthine (1.85 GBq/mmol) and 28-deoxy-D-[U-14C]glucose (8.5 GBq/mmol) were purchased from Amersham International, NSW, Australia. Isolation of poly(A+)-mRNA. Cells were harvested by centrifugation at 2500g for 20 min at 22–248C (Hall et al., 1993). The pelleted cells were washed twice with PBS and resuspended in 10 ml RNA Isolator (Genosys) per 2 3 109 cells. Total RNA was prepared following the protocol recommended by the manufacturer. Total RNA was precipitated with ice-cold 75% ethanol (v/v in diethylpyrocarbonate (DEPC)treated water) and then dissolved in DEPC-treated water (Ambion). Poly(A)+-mRNA was purified by oligo(dT)-cellulose chromatography using the Poly(A)Pure mRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. After precipitation with ethanol (70%) the RNA was dissolved in DEPC-treated water to a final concentration of 1 mg/ml and stored at 2708C prior to microinjection. RNA gel electrophoresis. The integrity of the isolated poly(A)+mRNA (5 mg per lane) was determined by electrophoresis on a 1.0 % agarose gel containing 2.2 M formaldehyde (Sambrook et al. 1989). Ethidium bromide (18.0 mg/ml)-labeled fractions including RNA size markers (Gibco BRL) were resolved by electrophoresis at 228C at 5 V/cm for 1.5 h. Microinjection of mRNA into oocytes. Approximately 50 nl
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poly(A)+-mRNA (1 mg/ml DEPC-water) or 50 nl DEPC-water was injected into defolliculated Stage V-VI X. laevis oocytes (Colman 1984) using a semiautomated microinjector (World Precision Instruments). Oocytes were maintained at 198C for 3–5 days in MBM (Colman 1984) with a daily change of medium. Oocyte uptake assays. For uptake assays, groups of 15–30 oocytes per condition were incubated for 120 min (unless otherwise stated) at 228C in MBM containing 15 mM [8-14C]D-adenosine, 15 mM [U14 C]hypoxanthine or 50 mM 28-deoxy-D-[U-14C]glucose. The reaction was terminated by removing oocytes from the incubation medium and rapidly washing 12 times in an excess of ice-cold MBM. Individual oocytes were transferred to 1.5 ml microfuge tubes, resuspended in 1 ml Optiphase Supermix scintillant (Wallac), and counted in a Wallac 1450 Microbeta Plus scintillation counter. Nucleoside and nucleobase metabolism studies. To ascertain that the expressed uptake of adenosine and hypoxanthine observed in these experiments was due to transport and not to subsequent metabolism, injected oocytes were incubated with either 15 mM [8-14C]D-adenosine or [U-14C]hypoxanthine for 120 min at 228C and the reaction was stopped as described above. Groups of 20 oocytes per condition were prepared for thin-layer chromatographic analysis. Metabolites associated with oocyte supernatants were analysed as described previously (Upston and Gero 1995). In both cases, approximately 93.0% of the radioactivity associated with oocytes incubated with [14C]adenosine or [14C]hypoxanthine remained in the form of [14C]adenosine and [14C]hypoxanthine, respectively. Data analysis. Data from oocyte uptake studies were not normally distributed and therefore were analysed using the nonparametric MannWhitney U test in SYSTAT (v5.2, SYSTAT Corp., U.S.A.).
RESULTS
Expression of adenosine and hypoxanthine transport in X. laevis oocytes injected with C. luciliae mRNA. Poly(A)+mRNA isolated from C. luciliae cultured under purinereplete conditions (as described under Materials and Methods) was microinjected into X. laevis oocytes; control oocytes were injected with DEPC-treated water. Each group contained approximately 30 individual oocytes. After 3 days at 198C, individual oocytes from each group were assayed for transport of adenosine (15 mM) and hypoxanthine (15 mM) over time periods from 15 s to 120 min (Fig. 1a and b, respectively). For mRNA-injected oocytes, uptake of adenosine and hypoxanthine over the time intervals used was significantly greater than that in water-injected control oocytes. The minor uptake in the water-injected oocytes confirmed previous reports that endogenous purine transporters of Xenopus oocytes have little activity (Huang et al., 1993), allowing studies of exogenous nucleoside/base transporters. As shown in Fig. 1, in this particular experiment, after 120 min of incubation, adenosine (Fig. 1a) and hypoxanthine (Fig. 1b) uptake in mRNA-injected oocytes
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FIG. 1. Time courses for transport of radiolabeled adenosine and hypoxanthine into X. laevis oocytes injected with C. luciliae poly(A)+mRNA. After 3 days injection, oocytes were incubated with (a) 15 mM [14C]D-adenosine or (b) 15 mM [14C]hypoxanthine and assayed for uptake of each substrate. Open circles represent mRNA-injected oocytes and closed circles represent the mean of water-injected control oocytes. Each time point represents uptake by a minimum of 10 individual oocytes including those which showed no expression. Differences between mRNA and water-injected oocytes were statistically significant at (a) 60 min (P , 0.002) and 120 min (P , 0.001), and at (b) 30 min (P , 0.001), 60 min (P , 0.036), and 120 min (P , 0.001).
was 13- and 8-fold greater than that in water-injected controls, respectively. Time points of only 120 min were used in all subsequent uptake assays. A second representative experiment of adenosine uptake but showing only transport at 120 min into Xenopus oocytes injected with mRNA isolated from purine-replete C. luciliae is shown in Fig. 2a. Adenosine uptake was increased 9-fold over water-injected oocytes. However, over the course of this work the mean adenosine influx calculated, using seven
different batches of mRNA isolated from purine-replete C. luciliae, and using seven separate batches of oocytes was 2.9 6 0.5 pmol/oocyte/120 min. This represents a mean 9-fold increase in uptake of adenosine into oocytes injected with mRNA from purine-replete cells, compared to waterinjected oocytes. Expression of adenosine transport in X. laevis oocytes using mRNA from purine-starved C. luciliae. Injection of poly(A)+-mRNA isolated from purine-starved C. luciliae
FIG. 2. Adenosine uptake into X. laevis oocytes injected with poly(A)+-mRNA obtained from purine-replete or -starved cells. Uptake of 15 mM [14C]D-adenosine into X. laevis oocytes injected with C. luciliae poly(A)+-mRNA isolated from cells grown in purine (a)-replete or (b)-starved medium as described under Materials and Methods. Control oocytes were injected with DEPC-treated water. Results represent one of at least five independent experiments for each condition. Data, represented by box plots (McGill et al., 1978), are median values (centre horizontal line) with notches indicating 95% confidence intervals, outer horizontal bars limiting the interquartile range (IQR) and whiskers representing the spread of the data (maxima to minima). Outliers (*) are defined as 1.5–3.0 3 IQR. Differences between experimental (RNA-injected) and control (waterinjected) groups were highly significant (P , 0.001).
184 (as described under Materials and Methods) into X. laevis oocytes assayed at 120 min (Fig. 2b) resulted in a mean 9-fold increase in adenosine (15 mM) uptake compared to water-injected controls. In five subsequent independent experiments using different batches of mRNA all from purinestarved cells, values for adenosine uptake into mRNA-injected oocytes ranged from 0.73 to 4.66 pmol/oocyte/120 min compared to 0.08 to 0.82 pmol/oocyte/120 min for oocytes injected with DEPC-treated water. In each experiment, the enhanced uptake of adenosine was highly statistically significant (P , 0.001). Overall, there was a mean 8fold increase in adenosine uptake in mRNA-injected compared to water-injected oocytes. Expression of hypoxanthine transport in X. laevis oocytes using mRNA from both purine-replete and -starved C. luciliae. Similar to the results obtained for adenosine uptake, transport of the purine nucleobase hypoxanthine (15 mM) into mRNA-injected oocytes from either purine-replete (Fig. 3a) or purine-starved (Fig. 3b) cells was not significantly different. In five separate experiments using different batches of mRNA isolated from purine-replete cells a 2.6 6 0.3 (mean 6 SEM)-fold increase in hypoxanthine uptake compared to water-injected controls was observed. In a further seven experiments, oocytes injected with mRNA isolated from purine-starved cells resulted in a 3.1 6 0.5 (mean 6 SEM)-fold increase in hypoxanthine influx compared with water-injected controls. C. luciliae responds in culture to purine deficiency by dramatically increasing transport of nucleosides and nucleobases (Alleman and Gottlieb 1996; Hall et al. 1996; Day and Gero 1997). Under purine-starved conditions, uptake of adenosine and hypoxanthine by C. luciliae in vitro was
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6- and 4-fold greater than in purine-replete cells (Fig. 4a). This phenomenon was specific for purine uptake. There was no corresponding increase for either glucose transport, using the nonmetabolisable glucose analogue 28-deoxy-D-glucose (28DOG), (Fig. 4a), or amino acid transport (Day and Gero 1997). Uptakes of adenosine, hypoxanthine, and 28DOG in oocytes injected with purine-replete and purine-starved mRNA is shown in Fig. 4b. Adenosine uptake into oocytes injected with purine-replete C. luciliae mRNA was not significantly different (P 5 0.375) to adenosine uptake into oocytes injected with purine-starved C. luciliae mRNA. Similarly for hypoxanthine, there was no significant difference in uptake in oocytes injected with mRNA obtained from either purine-replete or purine-starved cells (P 5 0.124). Expression of 28DOG uptake in oocytes injected with C. luciliae mRNA from purine-replete and -starved cells indicated that levels of expression of glucose transport were also not significantly different between both groups, consistent with findings in whole organisms (Day and Gero 1997). The observed lack of differences in expression of adenosine, hypoxanthine, and glucose transporters in Xenopus oocytes from mRNA isolated from cells grown in purinereplete or purine-depleted medium suggested that similar levels of mRNA for those transporters were present under both environmental conditions. As C. luciliae poly(A)+mRNA was microinjected and therefore the concentration of the specific mRNA species for the respective transporters was likely to be at a very low concentration, it is unlikely that the system was saturated with mRNA (which would prevent detection of variable transcript levels). However, in order to confirm this assumption, pure cRNA encoding a T. brucei hexose transporter (THT1) (Bringaud and Baltz 1993)
FIG. 3. Hypoxanthine uptake into X. laevis oocytes injected with poly (A)+-mRNA obtained from purine-replete or -starved cells. Uptake of 15 mM [14C]hypoxanthine into X. laevis oocytes injected with C. luciliae poly(A)+-mRNA isolated from cells grown in purine (a)-replete or (b)starved medium as described under Materials and Methods. Control oocytes were injected with DEPC-treated water. Results represent one of at least five independent experiments for each condition. Data are represented as described in the legend to Fig. 2. Differences between mRNA and water-injected oocytes were highly statistically significant: (a) P , 0.001; (b) P 5 0.003.
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FIG. 5. Adenosine uptake into X. laevis oocytes injected with poly(A)+-mRNA isolated from purine-replete or -starved C. luciliae or THT1 cRNA. Oocytes were assayed for transport of 15 mM [14C]adenosine 3 days postinjection. Data represent mean values 6 SEM.
FIG. 4. Transport of adenosine, hypoxanthine, and 28-deoxy-Dglucose (28DOG) into purine-replete and -starved C. luciliae cells compared with uptake into X. laevis oocytes injected with poly(A)+mRNA isolated from purine-replete and -starved cells. (a) Transport of 1 mM [3H]adenosine, 1 mM [3H]hypoxanthine, and 0.5 mM [3H]28DOG into C. luciliae organisms grown under purine-replete and purine-starved conditions as previously described (Hall et al., 1996; Day and Gero, 1997). (b) Net uptake of 15 mM [14C]adenosine, 15 mM [14C]hypoxanthine, and 50 mM [14C]-28-deoxy-D-glucose into oocytes injected with C. luciliae poly(A)+-mRNA isolated from purine-replete and purine-starved cells. Net uptake was determined by subtracting uptake in water-injected oocytes from the total expressed uptake. Data represent mean values from a minimum of five experiments.
was microinjected and the oocytes assayed for 28DOG uptake. Approximately 200-fold greater activity in 28DOG uptake was observed in cRNA-injected oocytes compared to poly(A)+-mRNA-injected oocytes (data not shown), indicating that the Xenopus system was well below the level of saturation with respect to the amounts of poly(A)+-mRNA injected. Additionally, control experiments showed no expression of adenosine transport activity from THT1 cRNAinjected oocytes (Fig. 5), thus eliminating the possibility of nonspecific activation of endogenous transporters by microinjection of RNA.
Substrate specificity of the expressed adenosine transporter. To determine the substrate specificity of the expressed adenosine transporter, the influx of adenosine into mRNA-injected and water-injected oocytes was measured in the presence of a 10-fold excess of putative competitors. Of those tested only the adenosine analogue 28-deoxy-Dadenosine significantly inhibited adenosine uptake; an inhibition of 44% in purine-replete and 64% in purine-starved mRNA-injected oocytes was observed. Neither adenine or hypoxanthine had a significant competitive effect, nor did the purine nucleoside guanosine. L-Adenosine, the stereoisomer of D-adenosine, had no effect on the induced D-adenosine uptake, demonstrating the stereospecificity of the expressed transporter (Table I). The expressed hypoxanthine uptake was inhibited by adenine and deoxy-D-adenosine (Table I), suggesting that hypoxanthine, adenine, and deoxyD-adenosine are substrates for the same transporter, and that this transporter is distinguishable from the adenosine transporter.
DISCUSSION
The Xenopus expression system is able to successfully synthesise biologically active protein after injection of exogenous mRNA (Gurdon et al. 1971). Described here is the first example of the functional expression of C. luciliaeencoded transport proteins in Xenopus oocytes. Injection of poly(A)+-mRNA from purine-starved and purine-replete C.
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TABLE I Effect of Nucleosides and Nucleobases on the Induced Uptake of Adenosine and Hypoxanthine in Xenopus Oocytes Injected with C. luciliae mRNA % Inhibition Permeant D-Adenosine
Competitor
Replete
Deoxy-D-adenosine 44 6 3.6 (32) Hypoxanthine 18 6 5.3 (27) Adenine 9 6 3.7 (8) Guanosine 4 6 0.4 (12) L-Adenosine 4 6 3.3 (22)
Starved 64 13 11 10 0
6 12.4 (9) 6 6.6 (30) 6 2.0 (14) 6 6.8 (9) (22)
Hypoxanthine Adenine 60 6 11.4 (39) 74 6 8.8 (46) Deoxy-D-adenosine 53 6 14.2 (7) 56 6 16.4 (10) Note. Oocytes were microinjected with poly(A)+-mRNA isolated from C. luciliae grown in purine-replete or purine-starved medium as described under Materials and Methods. After 3 days incubation at 198C, injected oocytes were incubated in 15 mM [14C]D-adenosine or [14C]hypoxanthine in the presence of 150 mM unlabeled competitor for 120 min at 228C. Endogenous [14C]D-adenosine and [14C] hypoxanthine uptake (i.e., water-injected) was subtracted from the total expressed uptake to give the “induced uptake.” All values were expressed as percentage inhibition of the control, where the control represents transport in the absence of competitor. Data represent means 6 SEM with number of oocytes in parentheses.
luciliae resulted in the expression of (i) an adenosine transporter which was stereospecific and exhibited high affinity for the purine nucleoside 28deoxy-D-adenosine but not the purine nucleobases hypoxanthine and adenine and (ii) a hypoxanthine transporter that was specific for adenine and deoxy-D-adenosine. In previous studies with Crithidia cultured under purinestarved conditions, competitors of the adenosine transporter included deoxy-D-adenosine, whereas no competition was observed by L-adenosine (Hall et al. 1996), hypoxanthine, or adenine. These substrate specificities of the adenosine transporter in whole cells are in agreement with the substrate specificity of the adenosine transporter expressed in Xenopus oocytes. Similarly, hypoxanthine transport in C. luciliae grown under purine-starved conditions was effectively competed by both adenine and deoxy-D-adenosine, again consistent with the properties of the hypoxanthine transporter expressed in purine-starved mRNA-injected Xenopus oocytes. Interestingly, adenine and deoxy-D-adenosine effectively competed for hypoxanthine uptake in Xenopus oocytes injected with poly(A)+-mRNA from purine-replete cells. In contrast, in whole organisms grown in purine-replete medium, hypoxanthine entered the cells by simple diffusion
and was therefore refractory to competition (Day and Gero 1997). C. luciliae responds in culture in purine-deficient medium by dramatically increasing expression of transporters which mediate uptake of nucleosides and nucleobases (Alleman and Gottlieb 1996; Hall et al. 1996; Day and Gero 1997). Under purine-starved conditions, uptake of adenosine and hypoxanthine by C. luciliae was on average 6- and 8-fold greater than in purine-replete cells (Hall et al. 1996; Day and Gero 1997). In addition to the activation of specific transport proteins, the activity of a 38-nucleotidase/nuclease (38-NTase) ectoenzyme capable of hydrolysing extracellular nucleic acids and 38-ribonucleotides to 58-nucleotides and nucleosides, respectively, has been identified in several members of the Trypanosomatidae (Gottlieb 1985). No mammalian 38-NTase has been reported. Moreover, in L. donovani and C. luciliae, the enzyme was also regulated by the availability of purines in the medium (Alleman and Gottlieb 1990). Therefore, it appears that members of the Trypanosomatidae, including C. luciliae, have developed several novel purine salvage strategies to overcome possible environmental constraints. A comparison of the expression of adenosine and hypoxanthine transporters in Xenopus oocytes following microinjection of poly(A)+-mRNA isolated from purine-replete and purine-starved C. luciliae revealed that there were no significant differences in levels of expression between the two groups. This result was in direct contrast to observations in whole cells and suggested that the amount of RNA transcripts encoding these membrane transporters was independent of the level of purines in the extracellular environment. Functional expression of these transporters in Xenopus oocytes suggested that the level of regulation/activation of these transporters as observed in whole organisms was due to post-transcriptional events. This is in agreement with previous studies in trypanosomatids, which indicates that regulation of gene expression is largely under post-transcriptional control (Pays 1993). Northern analysis of C. luciliae mRNA to detect amounts of purine transporter message relative to transporter activity in purine-replete compared with purine-starved cells would show conclusively whether regulation occurred at the level of transcription. However, such experiments require specific nucleotide probes for these transporters which at present are not available for C. luciliae. Possible factors controlling the expression of purine transporters could be at the translational or post-translational level. Cycloheximide, an inhibitor of eukaryotic translation, inhibited the activation of adenosine and hypoxanthine transporters in cells grown in purine-deficient medium (Hall et
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al. 1996; Day and Gero 1997). Furthermore, the downregulation of activated purine transporters was blocked when cells were transferred to purine-replete medium containing cycloheximide (Hall et al. 1996). These results suggest that (i) new protein synthesis is required for the observed activation in transporter activity, and (ii) cycloheximide may inhibit the synthesis of proteins involved in the regulation of purine transporters. Interestingly, the diamine transporter of the related trypanosomatid, T. cruzi, is upregulated in response to low extracellular putrescine levels (Quesne and Fairlamb 1996). In addition, folate transport in L. major promastigotes is downregulated in cells grown in high external folate concentrations (Ellenberger and Beverley 1987). Hence, several distinct transporter systems of the Trypanosomatidae are regulated by the availability of specific nutrients in the external milieu. The molecular mechanisms involved in this regulation remain to be elucidated. The assembly of newly synthesised transporters to the Xenopus oocyte membrane has facilitated the functional characterisation of hexose transporters of several trypanosomatids including L. enrietti (Langford et al. 1994), L. donovani (Langford et al. 1995), and T. brucei (Bringaud and Baltz 1993; Barrett et al. 1995). Recently, the human erythrocyte es nucleoside transporter (Griffiths et al. 1997) was also characterised by expression in X. laevis oocytes. The successful application of the X. laevis in vivo translation system to C. luciliae is demonstrated and represents a novel strategy in which the purine transporters (and hence other nutrient transporters) of these parasites may be isolated and cloned. As purine salvage is fundamental to parasite survival, knowledge of the molecular mechanisms involved in the “salvage” process may be exploited to provide new chemotherapeutic strategies. The upregulation in purine transporter activity observed in C. luciliae represents an adaptive response by the parasite to limiting levels of essential nutrients (i.e., purines) in the environment. As purine salvage is fundamental to parasite survival, further research needs to be carried out to determine whether similar regulatory mechanisms occur in related parasites of public health importance.
ACKNOWLEDGMENTS
Sanjeev Krishna is a Wellcome Trust Senior Research Fellow in Clinical Science. Annette Gero is supported by the National Health and Medical Research Council of Australia and the UNDP/World Bank/ WHO Special Program for Research and Training in Tropical Diseases. We thank Dr. Charles Woodrow for glucose uptake assays in THT1
cRNA-injected oocytes. The plasmid encoding THT1 was a gift from F. Bringaud. The authors are grateful to Professor Kiaran Kirk and Dr. Michael Barrett for their support and interest in this work.
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subcellular localisation of a high-Km hexose transporter from Leishmania donovani. Biochemistry 34, 11814–11821. Langford, C. K., Little, B. M., Kavanaugh, M. P., and Landfear, S. M. 1994. Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enrietti. The Journal of Biological Chemistry 269, 17939–17943. McGill, R., Tukey, J. W., and Larsen, W. A. 1978. Variation in box plots. The American Statistician 32, 12–16. Pays, E. 1993. Of gene expression in Trypanosomatids. In “The Eukaryotic Genome: Organisation and Regulation” (P. M. A. Broda S. G. O., and P. F. G. Sims, Ed). Cambridge Univ. Press, Cambridge. Quesne, S. A., and Fairlamb, A. H. 1996. Regulation of a high affinity diamine transport system in Trypanosoma cruzi epimastigotes. Biochemical Journal 316, 481–486. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Extraction and purification of RNA. In “Molecular Cloning: A Laboratory Manual” (C. Nolan, Ed). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Huang, Q.-Q., Harvey, C. M., Paterson, A. R. P., Cass, C. E., and Young, J. D. 1993. Functional expression of Na+-dependent nucleoside transport systems of rat intestine in isolated oocytes of Xenopus laevis. The Journal of Biological Chemistry 268, 20316–20619.
Upston, J. M., and Gero, A. M. 1995. Parasite-induced permeation of nucleosides in Plasmodium falciparum malaria. Biochemica et Biophysica Acta 1236, 249–258.
Kidder, G. W., Dewey, V. C., and Nolan, L. L. 1978. Transport and accumulation of purine bases by Crithidia fasciculata. Journal of Cell Physiology 96, 165–170.
Vasudevan, G., Drew, M., Carter, N., Beverley, S., Ullman, B., and Landfear, S. 1997. Cloning the Leishmania donovani adenosinepyrimidine nucleoside transporter by genetic complementation. Molecular Parasitology Meeting VIII, Woods Hole.
Langford, C. K., Kavanaugh, M. P., Stenberg, P. E., Drew, M. E., Zhang, W., and Landfear, S. M. 1995. Functional expression and
Received 12 January 1998; accepted 11 May 1998
90, 181–188 (1998)
PR984320
Crithidia luciliae: Functional Expression of Nucleoside and Nucleobase Transporters in Xenopus laevis Oocytes
Simone T. Hall,*,1 Jeffrey I. Penny,† Annette M. Gero,* and Sanjeev Krishna† *School of Biochemistry and Molecular Genetics, The University of NSW, Sydney, NSW, 2052, Australia; and †Division of Infectious Diseases, St. George’s Hospital Medical School, London, SW17 ORE, U.K.
Hall, S. T., Penny, J. I., Gero, A. M., and Krishna, S. 1998. Crithidia luciliae: Functional expression of nucleoside and nucleobase transporters in Xenopus laevis oocytes. Experimental Parasitology 90, 181–188. The expression of purine-specific nucleoside and base transporters of Crithidia luciliae has been demonstrated in Xenopus laevis oocytes. Poly(A)+-mRNA from C. luciliae, cultured in either purine-replete or purine-starved conditions, was microinjected into X. laevis oocytes. For “purine-replete” mRNA, expression of adenosine and hypoxanthine uptake in microinjected X. laevis oocytes was increased on average 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with “purine-starved” mRNA was 8 and 3-fold above water-injected controls, respectively. Substrate competition indicated an adenosine/deoxyadenosine transporter and a separate base transporter specific for hypoxanthine. In contrast to C. luciliae in vivo, where the level of activity of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters (from both purine replete and deplete cultures) in X. laevis oocytes was independent of the extracellular purine concentration. These results may suggest that the presence of specific transporter message is independent of the extracellular purine content, indicating that the regulation of activation and expression of these transporters in C. luciliae may not be under transcriptional control. q 1998 Academic Press Index Descriptors and Abbreviations: Crithidia luciliae; Xenopus laevis; functional expression; adenosine transporter; hypoxanthine transporter; 38-nucleotidase/nuclease, 38-NTase (EC 3.1.3.6); DEPC, diethylpyrocarbonate; MBM, modified Barths’ medium; 28DOG, 28-deoxy-D-glucose; IQR, interquartile range.
1 To whom correspondence should be addressed. Fax: (612) 93851483. E-mail: [email protected].
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
INTRODUCTION
Parasitic protozoa lack a de novo purine biosynthetic pathway and hence depend on the salvage of purines for nucleic acid synthesis by specific transport proteins. The trypanosomatid Crithidia luciliae is closely related to the pathogenic trypanosomatids Leishmania spp. and Trypanosoma spp. Biochemical evidence indicates that C. luciliae possess at least three distinct purine transport systems. Two of these mediate uptake of purine nucleosides: one for adenosine and its analogues and one for guanosine, its analogues, and inosine (Hall et al. 1993). The third represents a separate base transporter with high affinity for hypoxanthine and adenine; although adenosine is also transported by this system (Day and Gero 1997). Purine transporters with similar substrate specificities have been characterised in L. braziliensis (Hansen et al. 1982), L. donovani promastigotes (Aronow et al. 1987), L. major promastigotes (Baer et al. 1992), T. brucei brucei procyclic and bloodstream forms (deKoning and Jarvis 1997), and C. fasciculata (Kidder et al. 1978). C. luciliae exhibits an unusual phenomenon in which the level of activity of transporters which mediate uptake of purine nucleosides and nucleobases is dependent upon the concentration of available purines in the culture medium. It has been observed that a dramatic increase in the level of adenosine, guanosine, and hypoxanthine transport occurs in C. luciliae grown in purine-deficient medium compared to cells grown in purine-replete medium (Alleman and Gottlieb 1996; Hall et al. 1996; Day and Gero 1997; Gero et al.
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182 1997). The increase in purine uptake observed under purinestarved conditions is reversed by the addition of extracellular purines to the medium (Hall et al. 1996; Day and Gero 1997). This nutrient-induced upregulation of nucleoside/ base transporters is a phenomenon not reported in any other protozoan parasite studied previously. The biological importance of these transporters is evident as protozoan parasites rely on purine salvage pathways to obtain essential nutrients from the extracellular milieu. Only recently the genes encoding two nucleoside transporters of the related parasite L. donovani have been isolated and sequenced (Carter et al. 1997; Vasudevan et al. 1997). We report here the functional expression in Xenopus oocytes of an adenosine and a hypoxanthine transporter from C. luciliae mRNA isolated from either purine-replete or purine-starved cells. These findings represent the first step in the characterisation of the genes encoding purine nucleoside and base transporters of C. luciliae.
MATERIALS AND METHODS
Cell culture. C. luciliae were grown aerobically in RPMI 1640 medium containing 25 mM NaHCO3 (pH 7.4) and supplemented with 2 mM L-glutamine and 10% (v/v) heat-inactivated foetal bovine serum (Trace Biosciences). Cultures were inoculated by the addition of 30 ml stationary phase cells ('2 3 107 cells/ml) to 300 ml complete medium, in 1-L glass culture flasks and maintained at 268C for 24 h for purine-replete cells or 72 h for purine-starved cells as previously described (Hall et al., 1996). Radiochemicals. [8-14C]D-Adenosine (1.85 [U-14C[GBq/mmol), [U-14C]hypoxanthine (1.85 GBq/mmol) and 28-deoxy-D-[U-14C]glucose (8.5 GBq/mmol) were purchased from Amersham International, NSW, Australia. Isolation of poly(A+)-mRNA. Cells were harvested by centrifugation at 2500g for 20 min at 22–248C (Hall et al., 1993). The pelleted cells were washed twice with PBS and resuspended in 10 ml RNA Isolator (Genosys) per 2 3 109 cells. Total RNA was prepared following the protocol recommended by the manufacturer. Total RNA was precipitated with ice-cold 75% ethanol (v/v in diethylpyrocarbonate (DEPC)treated water) and then dissolved in DEPC-treated water (Ambion). Poly(A)+-mRNA was purified by oligo(dT)-cellulose chromatography using the Poly(A)Pure mRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. After precipitation with ethanol (70%) the RNA was dissolved in DEPC-treated water to a final concentration of 1 mg/ml and stored at 2708C prior to microinjection. RNA gel electrophoresis. The integrity of the isolated poly(A)+mRNA (5 mg per lane) was determined by electrophoresis on a 1.0 % agarose gel containing 2.2 M formaldehyde (Sambrook et al. 1989). Ethidium bromide (18.0 mg/ml)-labeled fractions including RNA size markers (Gibco BRL) were resolved by electrophoresis at 228C at 5 V/cm for 1.5 h. Microinjection of mRNA into oocytes. Approximately 50 nl
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poly(A)+-mRNA (1 mg/ml DEPC-water) or 50 nl DEPC-water was injected into defolliculated Stage V-VI X. laevis oocytes (Colman 1984) using a semiautomated microinjector (World Precision Instruments). Oocytes were maintained at 198C for 3–5 days in MBM (Colman 1984) with a daily change of medium. Oocyte uptake assays. For uptake assays, groups of 15–30 oocytes per condition were incubated for 120 min (unless otherwise stated) at 228C in MBM containing 15 mM [8-14C]D-adenosine, 15 mM [U14 C]hypoxanthine or 50 mM 28-deoxy-D-[U-14C]glucose. The reaction was terminated by removing oocytes from the incubation medium and rapidly washing 12 times in an excess of ice-cold MBM. Individual oocytes were transferred to 1.5 ml microfuge tubes, resuspended in 1 ml Optiphase Supermix scintillant (Wallac), and counted in a Wallac 1450 Microbeta Plus scintillation counter. Nucleoside and nucleobase metabolism studies. To ascertain that the expressed uptake of adenosine and hypoxanthine observed in these experiments was due to transport and not to subsequent metabolism, injected oocytes were incubated with either 15 mM [8-14C]D-adenosine or [U-14C]hypoxanthine for 120 min at 228C and the reaction was stopped as described above. Groups of 20 oocytes per condition were prepared for thin-layer chromatographic analysis. Metabolites associated with oocyte supernatants were analysed as described previously (Upston and Gero 1995). In both cases, approximately 93.0% of the radioactivity associated with oocytes incubated with [14C]adenosine or [14C]hypoxanthine remained in the form of [14C]adenosine and [14C]hypoxanthine, respectively. Data analysis. Data from oocyte uptake studies were not normally distributed and therefore were analysed using the nonparametric MannWhitney U test in SYSTAT (v5.2, SYSTAT Corp., U.S.A.).
RESULTS
Expression of adenosine and hypoxanthine transport in X. laevis oocytes injected with C. luciliae mRNA. Poly(A)+mRNA isolated from C. luciliae cultured under purinereplete conditions (as described under Materials and Methods) was microinjected into X. laevis oocytes; control oocytes were injected with DEPC-treated water. Each group contained approximately 30 individual oocytes. After 3 days at 198C, individual oocytes from each group were assayed for transport of adenosine (15 mM) and hypoxanthine (15 mM) over time periods from 15 s to 120 min (Fig. 1a and b, respectively). For mRNA-injected oocytes, uptake of adenosine and hypoxanthine over the time intervals used was significantly greater than that in water-injected control oocytes. The minor uptake in the water-injected oocytes confirmed previous reports that endogenous purine transporters of Xenopus oocytes have little activity (Huang et al., 1993), allowing studies of exogenous nucleoside/base transporters. As shown in Fig. 1, in this particular experiment, after 120 min of incubation, adenosine (Fig. 1a) and hypoxanthine (Fig. 1b) uptake in mRNA-injected oocytes
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FIG. 1. Time courses for transport of radiolabeled adenosine and hypoxanthine into X. laevis oocytes injected with C. luciliae poly(A)+mRNA. After 3 days injection, oocytes were incubated with (a) 15 mM [14C]D-adenosine or (b) 15 mM [14C]hypoxanthine and assayed for uptake of each substrate. Open circles represent mRNA-injected oocytes and closed circles represent the mean of water-injected control oocytes. Each time point represents uptake by a minimum of 10 individual oocytes including those which showed no expression. Differences between mRNA and water-injected oocytes were statistically significant at (a) 60 min (P , 0.002) and 120 min (P , 0.001), and at (b) 30 min (P , 0.001), 60 min (P , 0.036), and 120 min (P , 0.001).
was 13- and 8-fold greater than that in water-injected controls, respectively. Time points of only 120 min were used in all subsequent uptake assays. A second representative experiment of adenosine uptake but showing only transport at 120 min into Xenopus oocytes injected with mRNA isolated from purine-replete C. luciliae is shown in Fig. 2a. Adenosine uptake was increased 9-fold over water-injected oocytes. However, over the course of this work the mean adenosine influx calculated, using seven
different batches of mRNA isolated from purine-replete C. luciliae, and using seven separate batches of oocytes was 2.9 6 0.5 pmol/oocyte/120 min. This represents a mean 9-fold increase in uptake of adenosine into oocytes injected with mRNA from purine-replete cells, compared to waterinjected oocytes. Expression of adenosine transport in X. laevis oocytes using mRNA from purine-starved C. luciliae. Injection of poly(A)+-mRNA isolated from purine-starved C. luciliae
FIG. 2. Adenosine uptake into X. laevis oocytes injected with poly(A)+-mRNA obtained from purine-replete or -starved cells. Uptake of 15 mM [14C]D-adenosine into X. laevis oocytes injected with C. luciliae poly(A)+-mRNA isolated from cells grown in purine (a)-replete or (b)-starved medium as described under Materials and Methods. Control oocytes were injected with DEPC-treated water. Results represent one of at least five independent experiments for each condition. Data, represented by box plots (McGill et al., 1978), are median values (centre horizontal line) with notches indicating 95% confidence intervals, outer horizontal bars limiting the interquartile range (IQR) and whiskers representing the spread of the data (maxima to minima). Outliers (*) are defined as 1.5–3.0 3 IQR. Differences between experimental (RNA-injected) and control (waterinjected) groups were highly significant (P , 0.001).
184 (as described under Materials and Methods) into X. laevis oocytes assayed at 120 min (Fig. 2b) resulted in a mean 9-fold increase in adenosine (15 mM) uptake compared to water-injected controls. In five subsequent independent experiments using different batches of mRNA all from purinestarved cells, values for adenosine uptake into mRNA-injected oocytes ranged from 0.73 to 4.66 pmol/oocyte/120 min compared to 0.08 to 0.82 pmol/oocyte/120 min for oocytes injected with DEPC-treated water. In each experiment, the enhanced uptake of adenosine was highly statistically significant (P , 0.001). Overall, there was a mean 8fold increase in adenosine uptake in mRNA-injected compared to water-injected oocytes. Expression of hypoxanthine transport in X. laevis oocytes using mRNA from both purine-replete and -starved C. luciliae. Similar to the results obtained for adenosine uptake, transport of the purine nucleobase hypoxanthine (15 mM) into mRNA-injected oocytes from either purine-replete (Fig. 3a) or purine-starved (Fig. 3b) cells was not significantly different. In five separate experiments using different batches of mRNA isolated from purine-replete cells a 2.6 6 0.3 (mean 6 SEM)-fold increase in hypoxanthine uptake compared to water-injected controls was observed. In a further seven experiments, oocytes injected with mRNA isolated from purine-starved cells resulted in a 3.1 6 0.5 (mean 6 SEM)-fold increase in hypoxanthine influx compared with water-injected controls. C. luciliae responds in culture to purine deficiency by dramatically increasing transport of nucleosides and nucleobases (Alleman and Gottlieb 1996; Hall et al. 1996; Day and Gero 1997). Under purine-starved conditions, uptake of adenosine and hypoxanthine by C. luciliae in vitro was
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6- and 4-fold greater than in purine-replete cells (Fig. 4a). This phenomenon was specific for purine uptake. There was no corresponding increase for either glucose transport, using the nonmetabolisable glucose analogue 28-deoxy-D-glucose (28DOG), (Fig. 4a), or amino acid transport (Day and Gero 1997). Uptakes of adenosine, hypoxanthine, and 28DOG in oocytes injected with purine-replete and purine-starved mRNA is shown in Fig. 4b. Adenosine uptake into oocytes injected with purine-replete C. luciliae mRNA was not significantly different (P 5 0.375) to adenosine uptake into oocytes injected with purine-starved C. luciliae mRNA. Similarly for hypoxanthine, there was no significant difference in uptake in oocytes injected with mRNA obtained from either purine-replete or purine-starved cells (P 5 0.124). Expression of 28DOG uptake in oocytes injected with C. luciliae mRNA from purine-replete and -starved cells indicated that levels of expression of glucose transport were also not significantly different between both groups, consistent with findings in whole organisms (Day and Gero 1997). The observed lack of differences in expression of adenosine, hypoxanthine, and glucose transporters in Xenopus oocytes from mRNA isolated from cells grown in purinereplete or purine-depleted medium suggested that similar levels of mRNA for those transporters were present under both environmental conditions. As C. luciliae poly(A)+mRNA was microinjected and therefore the concentration of the specific mRNA species for the respective transporters was likely to be at a very low concentration, it is unlikely that the system was saturated with mRNA (which would prevent detection of variable transcript levels). However, in order to confirm this assumption, pure cRNA encoding a T. brucei hexose transporter (THT1) (Bringaud and Baltz 1993)
FIG. 3. Hypoxanthine uptake into X. laevis oocytes injected with poly (A)+-mRNA obtained from purine-replete or -starved cells. Uptake of 15 mM [14C]hypoxanthine into X. laevis oocytes injected with C. luciliae poly(A)+-mRNA isolated from cells grown in purine (a)-replete or (b)starved medium as described under Materials and Methods. Control oocytes were injected with DEPC-treated water. Results represent one of at least five independent experiments for each condition. Data are represented as described in the legend to Fig. 2. Differences between mRNA and water-injected oocytes were highly statistically significant: (a) P , 0.001; (b) P 5 0.003.
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FIG. 5. Adenosine uptake into X. laevis oocytes injected with poly(A)+-mRNA isolated from purine-replete or -starved C. luciliae or THT1 cRNA. Oocytes were assayed for transport of 15 mM [14C]adenosine 3 days postinjection. Data represent mean values 6 SEM.
FIG. 4. Transport of adenosine, hypoxanthine, and 28-deoxy-Dglucose (28DOG) into purine-replete and -starved C. luciliae cells compared with uptake into X. laevis oocytes injected with poly(A)+mRNA isolated from purine-replete and -starved cells. (a) Transport of 1 mM [3H]adenosine, 1 mM [3H]hypoxanthine, and 0.5 mM [3H]28DOG into C. luciliae organisms grown under purine-replete and purine-starved conditions as previously described (Hall et al., 1996; Day and Gero, 1997). (b) Net uptake of 15 mM [14C]adenosine, 15 mM [14C]hypoxanthine, and 50 mM [14C]-28-deoxy-D-glucose into oocytes injected with C. luciliae poly(A)+-mRNA isolated from purine-replete and purine-starved cells. Net uptake was determined by subtracting uptake in water-injected oocytes from the total expressed uptake. Data represent mean values from a minimum of five experiments.
was microinjected and the oocytes assayed for 28DOG uptake. Approximately 200-fold greater activity in 28DOG uptake was observed in cRNA-injected oocytes compared to poly(A)+-mRNA-injected oocytes (data not shown), indicating that the Xenopus system was well below the level of saturation with respect to the amounts of poly(A)+-mRNA injected. Additionally, control experiments showed no expression of adenosine transport activity from THT1 cRNAinjected oocytes (Fig. 5), thus eliminating the possibility of nonspecific activation of endogenous transporters by microinjection of RNA.
Substrate specificity of the expressed adenosine transporter. To determine the substrate specificity of the expressed adenosine transporter, the influx of adenosine into mRNA-injected and water-injected oocytes was measured in the presence of a 10-fold excess of putative competitors. Of those tested only the adenosine analogue 28-deoxy-Dadenosine significantly inhibited adenosine uptake; an inhibition of 44% in purine-replete and 64% in purine-starved mRNA-injected oocytes was observed. Neither adenine or hypoxanthine had a significant competitive effect, nor did the purine nucleoside guanosine. L-Adenosine, the stereoisomer of D-adenosine, had no effect on the induced D-adenosine uptake, demonstrating the stereospecificity of the expressed transporter (Table I). The expressed hypoxanthine uptake was inhibited by adenine and deoxy-D-adenosine (Table I), suggesting that hypoxanthine, adenine, and deoxyD-adenosine are substrates for the same transporter, and that this transporter is distinguishable from the adenosine transporter.
DISCUSSION
The Xenopus expression system is able to successfully synthesise biologically active protein after injection of exogenous mRNA (Gurdon et al. 1971). Described here is the first example of the functional expression of C. luciliaeencoded transport proteins in Xenopus oocytes. Injection of poly(A)+-mRNA from purine-starved and purine-replete C.
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TABLE I Effect of Nucleosides and Nucleobases on the Induced Uptake of Adenosine and Hypoxanthine in Xenopus Oocytes Injected with C. luciliae mRNA % Inhibition Permeant D-Adenosine
Competitor
Replete
Deoxy-D-adenosine 44 6 3.6 (32) Hypoxanthine 18 6 5.3 (27) Adenine 9 6 3.7 (8) Guanosine 4 6 0.4 (12) L-Adenosine 4 6 3.3 (22)
Starved 64 13 11 10 0
6 12.4 (9) 6 6.6 (30) 6 2.0 (14) 6 6.8 (9) (22)
Hypoxanthine Adenine 60 6 11.4 (39) 74 6 8.8 (46) Deoxy-D-adenosine 53 6 14.2 (7) 56 6 16.4 (10) Note. Oocytes were microinjected with poly(A)+-mRNA isolated from C. luciliae grown in purine-replete or purine-starved medium as described under Materials and Methods. After 3 days incubation at 198C, injected oocytes were incubated in 15 mM [14C]D-adenosine or [14C]hypoxanthine in the presence of 150 mM unlabeled competitor for 120 min at 228C. Endogenous [14C]D-adenosine and [14C] hypoxanthine uptake (i.e., water-injected) was subtracted from the total expressed uptake to give the “induced uptake.” All values were expressed as percentage inhibition of the control, where the control represents transport in the absence of competitor. Data represent means 6 SEM with number of oocytes in parentheses.
luciliae resulted in the expression of (i) an adenosine transporter which was stereospecific and exhibited high affinity for the purine nucleoside 28deoxy-D-adenosine but not the purine nucleobases hypoxanthine and adenine and (ii) a hypoxanthine transporter that was specific for adenine and deoxy-D-adenosine. In previous studies with Crithidia cultured under purinestarved conditions, competitors of the adenosine transporter included deoxy-D-adenosine, whereas no competition was observed by L-adenosine (Hall et al. 1996), hypoxanthine, or adenine. These substrate specificities of the adenosine transporter in whole cells are in agreement with the substrate specificity of the adenosine transporter expressed in Xenopus oocytes. Similarly, hypoxanthine transport in C. luciliae grown under purine-starved conditions was effectively competed by both adenine and deoxy-D-adenosine, again consistent with the properties of the hypoxanthine transporter expressed in purine-starved mRNA-injected Xenopus oocytes. Interestingly, adenine and deoxy-D-adenosine effectively competed for hypoxanthine uptake in Xenopus oocytes injected with poly(A)+-mRNA from purine-replete cells. In contrast, in whole organisms grown in purine-replete medium, hypoxanthine entered the cells by simple diffusion
and was therefore refractory to competition (Day and Gero 1997). C. luciliae responds in culture in purine-deficient medium by dramatically increasing expression of transporters which mediate uptake of nucleosides and nucleobases (Alleman and Gottlieb 1996; Hall et al. 1996; Day and Gero 1997). Under purine-starved conditions, uptake of adenosine and hypoxanthine by C. luciliae was on average 6- and 8-fold greater than in purine-replete cells (Hall et al. 1996; Day and Gero 1997). In addition to the activation of specific transport proteins, the activity of a 38-nucleotidase/nuclease (38-NTase) ectoenzyme capable of hydrolysing extracellular nucleic acids and 38-ribonucleotides to 58-nucleotides and nucleosides, respectively, has been identified in several members of the Trypanosomatidae (Gottlieb 1985). No mammalian 38-NTase has been reported. Moreover, in L. donovani and C. luciliae, the enzyme was also regulated by the availability of purines in the medium (Alleman and Gottlieb 1990). Therefore, it appears that members of the Trypanosomatidae, including C. luciliae, have developed several novel purine salvage strategies to overcome possible environmental constraints. A comparison of the expression of adenosine and hypoxanthine transporters in Xenopus oocytes following microinjection of poly(A)+-mRNA isolated from purine-replete and purine-starved C. luciliae revealed that there were no significant differences in levels of expression between the two groups. This result was in direct contrast to observations in whole cells and suggested that the amount of RNA transcripts encoding these membrane transporters was independent of the level of purines in the extracellular environment. Functional expression of these transporters in Xenopus oocytes suggested that the level of regulation/activation of these transporters as observed in whole organisms was due to post-transcriptional events. This is in agreement with previous studies in trypanosomatids, which indicates that regulation of gene expression is largely under post-transcriptional control (Pays 1993). Northern analysis of C. luciliae mRNA to detect amounts of purine transporter message relative to transporter activity in purine-replete compared with purine-starved cells would show conclusively whether regulation occurred at the level of transcription. However, such experiments require specific nucleotide probes for these transporters which at present are not available for C. luciliae. Possible factors controlling the expression of purine transporters could be at the translational or post-translational level. Cycloheximide, an inhibitor of eukaryotic translation, inhibited the activation of adenosine and hypoxanthine transporters in cells grown in purine-deficient medium (Hall et
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al. 1996; Day and Gero 1997). Furthermore, the downregulation of activated purine transporters was blocked when cells were transferred to purine-replete medium containing cycloheximide (Hall et al. 1996). These results suggest that (i) new protein synthesis is required for the observed activation in transporter activity, and (ii) cycloheximide may inhibit the synthesis of proteins involved in the regulation of purine transporters. Interestingly, the diamine transporter of the related trypanosomatid, T. cruzi, is upregulated in response to low extracellular putrescine levels (Quesne and Fairlamb 1996). In addition, folate transport in L. major promastigotes is downregulated in cells grown in high external folate concentrations (Ellenberger and Beverley 1987). Hence, several distinct transporter systems of the Trypanosomatidae are regulated by the availability of specific nutrients in the external milieu. The molecular mechanisms involved in this regulation remain to be elucidated. The assembly of newly synthesised transporters to the Xenopus oocyte membrane has facilitated the functional characterisation of hexose transporters of several trypanosomatids including L. enrietti (Langford et al. 1994), L. donovani (Langford et al. 1995), and T. brucei (Bringaud and Baltz 1993; Barrett et al. 1995). Recently, the human erythrocyte es nucleoside transporter (Griffiths et al. 1997) was also characterised by expression in X. laevis oocytes. The successful application of the X. laevis in vivo translation system to C. luciliae is demonstrated and represents a novel strategy in which the purine transporters (and hence other nutrient transporters) of these parasites may be isolated and cloned. As purine salvage is fundamental to parasite survival, knowledge of the molecular mechanisms involved in the “salvage” process may be exploited to provide new chemotherapeutic strategies. The upregulation in purine transporter activity observed in C. luciliae represents an adaptive response by the parasite to limiting levels of essential nutrients (i.e., purines) in the environment. As purine salvage is fundamental to parasite survival, further research needs to be carried out to determine whether similar regulatory mechanisms occur in related parasites of public health importance.
ACKNOWLEDGMENTS
Sanjeev Krishna is a Wellcome Trust Senior Research Fellow in Clinical Science. Annette Gero is supported by the National Health and Medical Research Council of Australia and the UNDP/World Bank/ WHO Special Program for Research and Training in Tropical Diseases. We thank Dr. Charles Woodrow for glucose uptake assays in THT1
cRNA-injected oocytes. The plasmid encoding THT1 was a gift from F. Bringaud. The authors are grateful to Professor Kiaran Kirk and Dr. Michael Barrett for their support and interest in this work.
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Received 12 January 1998; accepted 11 May 1998