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Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei
Abstract
The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a Km of 9.4±2.8 μM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (Km=0.38±0.06 μM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5–15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (Km remained unchanged) and inhibited by cycloheximide (10 μM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (Km∼0.2 μM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (Km∼10 μM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.
... The free ribosides are mostly intermediate metabolites. Indeed, ribosides can cross the cell membrane, taking advantage of equilibrative nucleoside transporters, since they lack the negative charges localized on the phosphate groups present in ribotides, including nucleotides [7,8]. Thus, this property of ribosides allows their use as a carrier of nitrogenous bases in the circulation and across cell membranes. ...
... Protozoan parasites of the Trypanosoma genus are purine auxotrophic [20] and rely on the bases obtained from the host for the synthesis of the corresponding nucleotides [12]. To this end, purine nucleosides are imported into the parasite via neutral nucleoside transporters [8,21], where they either undergo direct phosphorylation via nucleoside kinases or N-glycosidic bond cleavage. Unlike other organisms, notably the mammalian hosts, trypanosomes do not display purine-specific NP activity. ...
... These results showed for the first time that inhibitors specific to the trypanosomal IAG-NHs can indeed act as trypanocidal compounds. The bioavailability of the compounds, namely the transport by equilibrative nucleoside transporters across the protozoan membrane [8,91], requires further optimization to provide a panel of compounds that can be further tested in in vivo models of infection. ...
Enzymes catalyzing the hydrolysis of the N-glycosidic bond in nucleosides and other ribosides (N-ribohydrolases, NHs) with diverse substrate specificities are found in all kingdoms of life. While the overall NH fold is highly conserved, limited substitutions and insertions can account for differences in substrate selection, catalytic efficiency, and distinct structural features. The NH structural module is also employed in monomeric proteins devoid of enzymatic activity with different physiological roles. The homo-oligomeric quaternary structure of active NHs parallels the different catalytic strategies used by each isozyme, while providing a buttressing effect to maintain the active site geometry and allow the conformational changes required for catalysis. The unique features of the NH catalytic strategy and structure make these proteins attractive targets for diverse therapeutic goals in different diseases.
... This stage-specific expression of adenosine transporters is correlated with the increased requirement for adenosine by the amastigotes as evidenced by the 50- fold increase in their adenosine kinase activity over the promastigotes (Looker et al., 1983). This developmental regulation of the capacity and specificity of purine transporters is not particular to Leishmania as it is now well documented in trypanosomes (de Koning et al., 2000b) and Plasmodium falciparum (Carter et al., 2000a). Adenosine is the best purine nucleoside taken up by Trypanosoma congolense and Trypanosoma brucei. ...
... while TbAT1 is expressed only in the blood form (de Koning et al., 1998). The activity of these transporters seems to be regulated by purine availability and growth cycle (de Koning et al., 2000b). Recently, it has been reported that TbAT1, in strain TREU 927 of T. brucei, is a member of a multigene family that encodes six similar but distinct transporters denominated TbNT2 to TbNT7 (Sanchez et al., 2002). ...
... The locus PfNT1 has been cloned and expressed (Carter et al., 2000a; Parker et al., 2000). Like in leishmania (Ghosh & Mukherjee, 2000) and in trypanosomes (de Koning et al., 2000b), the expression of PfNT1 is strongly regulated during the multiplication of the parasite erythro-cytic stage. The increase in PfNT1 expression during the life cycle is associated with the increased requirements and salvage of purines as well as the incorporation of adenosine into nucleic acids (Carter et al., 2000a). ...
... As expected, PYR6- 5 2/2 cells were unable to grow in this semi-defined medium without pyrimidines, and the trypanosome population rapidly declined after 24 h (Fig. 2A,B). In contrast, a shift to purine-free conditions only caused growth arrest after approximately 48 h (Fig. 2B), consistent with previous observations in procyclic T. brucei [38]. Evidently, any interruption in pyrimidine supply rapidly makes trypanosomes non-viable and we investigated how quickly the damage becomes irreversible (Fig. 2A), by adding back 100 mM uracil at various times after passage of PYR6-5 2/2 to HMI-9 -tmd . ...
... *, P,0.05; **, P,0.01. doi:10.1371/journal.pone.0058034.g007 of purines [38]. It can easily be speculated that kinetoplastid parasites, having evolved without the capacity to synthesise their own purines, must be relatively well-adapted to periods with relatively low purine availability, as demonstrated by the reversibility of purine starvation-induced growth arrest [38]. ...
... doi:10.1371/journal.pone.0058034.g007 of purines [38]. It can easily be speculated that kinetoplastid parasites, having evolved without the capacity to synthesise their own purines, must be relatively well-adapted to periods with relatively low purine availability, as demonstrated by the reversibility of purine starvation-induced growth arrest [38]. In contrast, they have not needed to develop a mechanism to cope with a prolonged dearth of pyrimidines, being able to make sufficient amounts themselves, and trypanosomes are therefore unable to recover from even short periods of pyrimidine starvation. ...
Background
African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.
Methodology/Principal Findings
Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.
Conclusions/Significance
Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.
... However, it remains unclea r as to whether this reflects biosynthesis of additiona l transporters or of regulatory proteins (or both), although all results so far are consistent with an increase d numbe r of active transporters in the plasma membrane. Significa ntly, changes in transport rates are the result of change s in V m a x , while the K m remains unchange d (De Koning et al. 2000). ...
... All the work on upregulation of purine transport in C. luciliae and C. fasciculata indicated a common, integrated respons e to purine starvation, activating 3¢ -nucleotidase , nucleobas e transporters and nucleoside transporters equally and on exactly the same time scale (Hall et al. 1996, Day and Gero 1997, De Koning et al. 2000. In addition, the activitie s of several enzymes of the purine metabolic pathways are all elevated during purine stress (Alleman and Gottlieb 1996). ...
... Response to purine stress appeared to be more diverse in procyclic T. b. brucei. While the P1 purine nucleoside transporter was upregulate d much as the C. luciliae transporters under the same conditions, the activity of the H1 purine nucleobas e and U1 uracil transporters were not affected (De Koning et al. 2000). Instead, a different nucleobas e transporte r activity, tentatively identifie d as the H2 transporter normally expressed only in bloodstrea m forms (De Koning and Jarvis 1997b), was observed. ...
Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members fromarchea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.
... Indeed, depletion of extracellular purines triggers a substantial upregulation in nucleoside uptake in the parasitic protozoa, Crithidia spp. and Trypanosoma brucei (de Koning et al., 2000, Alleman & Gottlieb, 1996, Hall et al., 1996, Liu et al., 2005). While these previous studies suggest that nucleoside transport is intimately linked to the extracellular purine milieu, little is known about the molecular mechanisms governing these changes. ...
... These changes in purine uptake were also accompanied by an upregulation in the activities and concomitant changes at the protein level for the purine salvage enzymes, HGPRT, APRT, and XPRT, suggesting that purine metabolism is globally remodeled in response to purine starvation. The adaptive response to purine starvation in L. donovani is similar to that described previously in other trypanosomatids (de Koning et al., 2000, Alleman & Gottlieb, 1996, Hall et al., 1996, Liu et al., 2005, Gottlieb, 1985, Sopwith et al., 2002, Yamage et al., 2000, Yamage et al., 2007), where increased nucleoside transport and/or purine salvage enzyme activities were observed under conditions of prolonged purine withdrawal. This intimates that all trypanosomatids possess a common mechanism for adapting to purine starvation and perhaps to any nutritional stress. ...
... Our results, together with those in the accompanying manuscript from Ortiz et al., suggest that Leishmania have evolved a comprehensive mechanism to adapt to purine stress that involves the augmentation of endogenous purine transport proteins as well as purine salvage components. These investigations, together with studies on several other trypanosomatids (Alleman & Gottlieb, 1996, de Koning et al., 2000, Hall et al., 1996, Liu et al., 2005) and a variety of eukaryotic cells (Chaudary et al., 2004, Coe et al., 2002), indicate that the translational control of purine salvage components is modulated by the extracellular milieu and ultimately, may prove pivotal in maintaining intracellular purine homeostasis and the energy balance of the cell under conditions of cellular stress.Table S1 of the Supporting information) and for generating the pSNAR-HA-LdNT2-RV episome were purchased from Integrated DNA Technologies (Coralville, IA). The mouse living colors A.v. monoclonal JL-8 GFP antibody was purchased from BD Biosciences (Palo Alto, CA), mouse monoclonal anti-α–tubulin DM1A was purchased from EMD Biosciences (La Jolla, CA), and the mouse monoclonal antihuman influenza hemagglutinin (HA) antibody was obtained from Sigma-Aldrich. ...
Starvation of Leishmania donovani parasites for purines leads to a rapid amplification in purine nucleobase and nucleoside transport. Studies with nucleoside transport-deficient L. donovani indicate that this phenomenon is mediated by the nucleoside transporters LdNT1 and LdNT2, as well as by the purine nucleobase transporter LdNT3. The escalation in nucleoside transport cannot be ascribed to an increase in either LdNT1 or LdNT2 mRNA. However, Western analyses on parasites expressing epitope-tagged LdNT2 revealed a marked upregulation in transporter protein at the cell surface. Kinetic investigations of LdNT1 and LdNT2 activities from purine-replete and purine-starved cells indicated that both transporters exhibited significant increases in V(max) for their ligands under conditions of purine-depletion, although neither transporter displayed an altered affinity for its respective ligands. Concomitant with the increase in purine nucleoside and nucleobase transport, the purine salvage enzymes HGPRT, XPRT and APRT were also upregulated, suggesting that under conditions where purines are limiting, Leishmania parasites remodel their purine metabolic pathway to maximize salvage. Moreover, qRT-PCR analyses coupled with cycloheximide inhibition studies suggest that the underlying molecular mechanism for this augmentation in purine salvage occurs post-transcriptionally and is reliant on de novo protein synthesis.
... However, it remains unclea r as to whether this reflects biosynthesis of additiona l transporters or of regulatory proteins (or both), although all results so far are consistent with an increase d numbe r of active transporters in the plasma membrane. Significa ntly, changes in transport rates are the result of change s in V m a x , while the K m remains unchange d (De Koning et al. 2000). ...
... All the work on upregulation of purine transport in C. luciliae and C. fasciculata indicated a common, integrated respons e to purine starvation, activating 3¢ -nucleotidase , nucleobas e transporters and nucleoside transporters equally and on exactly the same time scale (Hall et al. 1996, Day and Gero 1997, De Koning et al. 2000. In addition, the activitie s of several enzymes of the purine metabolic pathways are all elevated during purine stress (Alleman and Gottlieb 1996). ...
... Response to purine stress appeared to be more diverse in procyclic T. b. brucei. While the P1 purine nucleoside transporter was upregulate d much as the C. luciliae transporters under the same conditions, the activity of the H1 purine nucleobas e and U1 uracil transporters were not affected (De Koning et al. 2000). Instead, a different nucleobas e transporte r activity, tentatively identifie d as the H2 transporter normally expressed only in bloodstrea m forms (De Koning and Jarvis 1997b), was observed. ...
Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.
... This stage-specific expression of adenosine transporters is correlated with the increased requirement for adenosine by the amastigotes as evidenced by the 50fold increase in their adenosine kinase activity over the promastigotes (Looker et al., 1983). This developmental regulation of the capacity and specificity of purine transporters is not particular to Leishmania as it is now well documented in trypanosomes (de Koning et al., 2000b) and Plasmodium falciparum (Carter et al., 2000a). ...
... while TbAT1 is expressed only in the blood form (de Koning et al., 1998). The activity of these transporters seems to be regulated by purine availability and growth cycle (de Koning et al., 2000b). ...
... The locus PfNT1 has been cloned and expressed (Carter et al., 2000a;Parker et al., 2000). Like in leishmania (Ghosh & Mukherjee, 2000) and in trypanosomes (de Koning et al., 2000b), the expression of PfNT1 is strongly regulated during the multiplication of the parasite erythro-cytic stage. The increase in PfNT1 expression during the life cycle is associated with the increased requirements and salvage of purines as well as the incorporation of adenosine into nucleic acids (Carter et al., 2000a). ...
Parasites are responsible for a wide variety of infectious diseases in human as well as in domestic and wild animals, causing an enormous health and economical blight. Current containment strategies are not entirely successful and parasitic infections are on the rise. In the absence of availability of antiparasitic vaccines, chemotherapy remains the mainstay for the treatment of most parasitic diseases. However, there is an urgent need for new drugs to prevent or combat some major parasitic infections because of lack of a single effective approach for controlling the parasites (e.g., trypanosomiasis) or because some serious parasitic infections developed resistance to presently available drugs (e.g., malaria). The rational design of a drug is usually based on biochemical and physiological differences between pathogens and host. Some of the most striking differences between parasites and their mammalian host are found in purine metabolism. Purine nucleotides can be synthesized by the de novo and/or the so-called "salvage" pathways. Unlike their mammalian host, most parasites studied lack the pathways for de novo purine biosynthesis and rely on the salvage pathways to meet their purine demands. Moreover, because of the great phylogenic separation between the host and the parasite, there are in some cases sufficient distinctions between corresponding enzymes of the purine salvage from the host and the parasite that can be exploited to design specific inhibitors or "subversive substrates" for the parasitic enzymes. Furthermore, the specificities of purine transport, the first step in purine salvage, diverge significantly between parasites and their mammalian host. This review highlights the unique transporters and enzymes responsible for the salvage of purines in parasites that could constitute excellent potential targets for the design of safe and effective antiparasitic drugs.
... 113 This confirms an older observation that hypoxanthine transport rates are increased during log phase and purine starvation response in procyclic trypanosomes. 114 Differently, ADET1 seems to be expressed by both T. brucei BSF and PCF and was probably not noticed earlier because none of the other studies of nucleobase transport in trypanosomes used [ 3 H]-adenine as permeant. 61,110 Interestingly, although the K m values determined in PCF and in BSF differ slightly, the activity for ADET1 is four times higher in PCF than in BSF, possibly indicating a higher demand of PCF for adenine, at least under standard axenic conditions. ...
... 61,110 Interestingly, although the K m values determined in PCF and in BSF differ slightly, the activity for ADET1 is four times higher in PCF than in BSF, possibly indicating a higher demand of PCF for adenine, at least under standard axenic conditions. 61,110 It has been demonstrated that the extracellular concentration of purines may alter the level of expression of purine transporters in T. brucei, 61,113,114 but this is unlikely to be the case for ADET1 as adenine is absent in both SDM-79 (for PCF) and HMI-9 (for BSF) media used for in vitro culture. ...
Purines and pyrimidines are essential nutrients for any cell. Most organisms are able to synthesize their own purines and pyrimidines, but this ability was lost in protozoans that adapted to parasitism, leading to a great diversification in transporter activities in these organisms, especially for the acquisition of amino acids and nucleosides from their hosts throughout their life cycles. Many of these transporters have been shown to have sufficiently different substrate affinities from mammalian transporters, making them good carriers for therapeutic agents. In this review, we summarize the knowledge obtained on purine and pyrimidine activities identified in protozoan parasites to date and discuss their importance for the survival of these parasites and as drug carriers, as well as the perspectives of developments in the field.
... Consequently, these parasites obligatorily rely on purine salvage from the host milieu in order to survive and proliferate (Marr et al., 1978, Hassan & Coombs, 1985, LaFon et al., 1982. Recent studies have demonstrated that Leishmania are capable of sensing the levels of purine in the extracellular environment (de Koning et al., 2000, Carter et al., 2010. In response to purine starvation, L. donovani rapidly upregulates purine salvage machinery including the plasma membrane nucleoside transporters, LdNT1 and LdNT2, and the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase (HGPRT), xanthine phosphoribosyltransferase (XPRT), and adenine phosphoribosyltransferase (APRT) as a strategy to maintain intracellular purine nucleotide levels (de Koning et al., 2000, Carter et al., 2010. ...
... Recent studies have demonstrated that Leishmania are capable of sensing the levels of purine in the extracellular environment (de Koning et al., 2000, Carter et al., 2010. In response to purine starvation, L. donovani rapidly upregulates purine salvage machinery including the plasma membrane nucleoside transporters, LdNT1 and LdNT2, and the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase (HGPRT), xanthine phosphoribosyltransferase (XPRT), and adenine phosphoribosyltransferase (APRT) as a strategy to maintain intracellular purine nucleotide levels (de Koning et al., 2000, Carter et al., 2010. ...
The Leishmania guanosine 5'-monophosphate reductase (GMPR) and inosine 5'-monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function in maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine-β-synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in E. coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH-dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the Km values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP Km value 10-fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools. This article is protected by copyright. All rights reserved.
... A separate study of BF trypanosomes also produced evidence for a distinct permease for S-adenosylmethionine (24). Purine transporters have also been studied with related Kinetoplastid parasites, including Crithidia fasciculata (21), Crithidia luciliae (26), and Trypanosoma cruzi (22). ...
... Initial studies by two groups (2, 25) using C. luciliae revealed that the rates of purine nucleoside and nucleobase uptake were dramatically upregulated by purine starvation and by the stage of the growth cycle. Subsequent studies demonstrated that the CfNT1 and CfNT2 nucleoside permease activities of C. fasciculata were robustly expressed during mid-logarithmic growth but were dramatically downregulated during the stationary phase (21). Furthermore, permease activity was stimulated 5-to 15-fold in purinedepleted medium. ...
... Previous work on adenosine transporter CfNT1 and inosine-guanosine transporter CfNT2 in C. fasciculata suggested the existence of multiple adenosine-transporting ENTs in this organism [9]. In addition, an adenosine uptake activity that was partially inhibited by cytidine was measured in whole Crithidia cells [9,10], but CfNT1-dependent adenosine transport was not inhibited in this way [9], suggesting biochemical differences might exist between CfNT1 and the unidentified adenosine transporter(s). ...
... CfNT1 expressed in L. donovani exhibited a significantly higher K m value than that of CfAT1 (59.0 ± 10 µM, n = 3; Figure 2B). Like CfAT1, CfNT1 showed high specificity for adenosine when expressed in L. donovani (Fig. 2D), suggesting that neither CfNT1 nor CfAT1 is responsible for the cytidine-inhibitable adenosine uptake observed in whole C. fasciculata cells [9,10]. ...
Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.
... These data confirm that the ATP hydrolysis stimulated by Mg 2+ is catalyzed by an authentic Mg-dependent ecto-ATPase. It is well known that trypanosomatids of the genus Trypanosoma are unable to synthesize purines de novo, and thus are dependent on exogenous sources of these essential nutrients (De Koning et al. 2000;Berreˆdo-Pinho et al. 2001). Extracellular ATP and its degradation products ADP, AMP and adenosine are normal components of the extracellular milieu. ...
... T. cruzi, as well as L. amazonensis, are pathogens which cannot synthesize purines de novo (De Koning et al. 2000;Berreˆdo-Pinho et al. 2001). It has been Fig. 8 Mg 2+ -dependent ecto-ATPase activity of different stages of T. cruzi. ...
In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42±0.31 nmol Pi/h×108 cells). ATP hydrolysis was stimulated by MgCl2, and the Mg-dependent ecto-ATPase activity was 27.15±2.91 nmol Pi/h×108 cells. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The apparent K
m for Mg-ATP2− was 0.61 mM, and free Mg2+ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4′.diisothiocyanostylbene 2′-2′-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg2+-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.
... In the presence of guanine as the sole purine in the medium, all trypanosome lines were much more sensitive to RBV probably due to an increased uptake in the absence of hypoxanthine. Indeed, an upregulation of the nucleoside P1 transporter, responsible for the uptake of ribavirin 36 , has been documented in T. brucei in response to purine stress 37 . Comparing cell lines, the degree of sensitivity to RBV was similar to the observed in a medium with only hypoxanthine; cells lacking ITPA were more resistant to RBV suggesting that accumulation ...
Inosine triphosphate pyrophosphatases (ITPases) are ubiquitous house-cleaning enzymes that specifically recognize deaminated purine nucleotides and catalyze their hydrolytic cleavage. In this work, we have characterized the Trypanosoma brucei ITPase ortholog (TbITPA). Recombinant TbITPA efficiently hydrolyzes (deoxy)ITP and XTP nucleotides into their respective monophosphate form. Immunolocalization analysis performed in bloodstream forms suggests that the primary role of TbITPA is the exclusion of deaminated purines from the cytosolic nucleoside triphosphate pools. Even though ITPA-knockout bloodstream parasites are viable, they are more sensitive to inhibition of IMP dehydrogenase with mycophenolic acid, likely due to an expansion of IMP, the ITP precursor. On the other hand, TbITPA can also hydrolyze the activated form of the antiviral ribavirin although in this case, the absence of ITPase activity in the cell confers protection against this nucleoside analog. This unexpected phenotype is dependant on purine availability and can be explained by the fact that ribavirin monophosphate, the reaction product generated by TbITPA, is a potent inhibitor of trypanosomal IMP dehydrogenase and GMP reductase. In summary, the present study constitutes the first report on a protozoan inosine triphosphate pyrophosphatase involved in the removal of harmful deaminated nucleotides from the cytosolic pool.
... They express a plethora of transporters, many of them developmentally regulated, to cope with the fluctuations in purine concentrations they likely encounter through their life cycles (27). Purine starvation in trypanosomatids results in the upregulation of nucleoside transport activity and a deep remodelling of purine metabolic pathways (24,28), and there is strong evidence that purines themselves act as powerful signals that trigger the metabolic adaptations necessary for cell survival when extracellular purines are scarce (29). Indeed, in Leishmania purine starvation activates a gene expression program that governs a developmental transition from promastigote to metacyclic cells, which are responsible for the initiation of infection in the mammalian host (30). ...
Post-transcriptional regulation of gene expression is particularly important in trypanosomatid protozoa. RNA-binding proteins (RBPs) regulate mRNA stability and translation, yet information about how RBPs are able to link environmental cues to post-transcriptional control is scarce. In Trypanosoma brucei, we have previously characterized a short RNA stem-loop cis-element (PuRE, Purine Responsive Element) within the 3′-UTR of the NT8 nucleobase transporter mRNA that is necessary and sufficient to confer a strong repression of gene expression in response to purines. In this study, we have identified a protein complex composed of two RNA-binding proteins (PuREBP1 and PuREBP2) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNA interference results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression.
... Incorporation of 32 P i into the cells was measured using a scintillation counter. To obtain the blank uptake values, parasites were exposed to a non-radioactive P i reaction mixture and then kept on ice for 60 min [81]. To determine the effect of pH on P i uptake, the same buffer described above was used, with the addition of 10 mM HEPES/MES/Tris at various pH values. ...
Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (P i ) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires P i from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous P i by exploiting a gradient of Na ⁺ ions to drive P i uptake across the plasma membrane. The Na ⁺ -dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na ⁺ -H ⁺ -ATPase. Toxoplasma expresses one transmembrane P i transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon P i limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing P i and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for P i supply for Toxoplasma and also for protection against osmotic stresses.
... The mixture containing the 32 P i taken up by the cells was then transferred to a scintillation vial containing 9.0 ml of scintillation fluid. Blank uptake values were obtained by exposing the cells to the reaction mixture and keeping them on ice during the 32 P i uptake reaction (de Koning et al., 2000). ...
The model yeast Saccharomyces cerevisiae elicits a transcriptional response to phosphate (Pi) depletion. To determine the origins of the phosphate response (PHO) system, we bioinformatically identified putative PHO components in the predicted proteomes of diverse fungi. Our results suggest that the PHO system is ancient; however, components have been expanded or lost in different fungal lineages. To show that a similar physiological response is present in deeply-diverging fungi we examined the transcriptional and physiological response of PHO genes to Pidepletion in the blastocladiomycete Blastocladiella emersonii. Our physiological experiments indicate that B. emersonii relies solely on high-affinity Na+-independent Pho84-like transporters. In response to Pidepletion, BePho84 paralogues were 4-8-fold transcriptionally upregulated, whereas several other PHO homologues like phosphatases and vacuolar transporter chaperone (VTC) complex components show 2-3-fold transcriptional upregulation. Since Pihas been shown to be important during the development of B. emersonii, we sought to determine if PHO genes are differentially regulated at different lifecycle stages. We demonstrate that a similar set of PHO transporters and phosphatases are upregulated at key points during B. emersonii development. Surprisingly, some genes upregulated during Pidepletion, including VTC components, are repressed at these key stages of development indicating that PHO genes are regulated by different pathways in different developmental and environmental situations. Overall, our findings indicate that a complex PHO network existed in the ancient branches of the fungi, persists in diverse extant fungi, and that this ancient network is likely to be involved in development and cell cycle regulation.
... If we consider the fact that the commonly used culture medium for bloodstream forms, HMI-9, contains 1 mM hypoxanthine, and that the transport of purines in trypanosomatids is protondependent [4][5][6], and coupled to a plasma membrane H + -ATPase to avoid cytoplasmic acidification [4,5], it is unsurprising that the cells drastically reduce the level of expression of such high-affinity transporters, in order to avoid unnecessarily high levels of hypoxanthine uptake. Indeed, it has previously been established that high affinity hypoxanthine (and adenosine) uptake in T. brucei bloodstream forms is correlated to the extracellular purine concentration and the cell cycle [59], as is the case for purine transport in the related parasite Crithidia luciliae [60]. ...
The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters - the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol(107 cells)-1s-1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol(107 cells)-1 s-1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family.
... The mixture containing the 32 P i taken up by the cells was then transferred (on a filter paper) to a scintillation vial containing 9.0 ml of scintillation fluid. Blank values of uptake were obtained by exposing the cells to the reaction mixture and keeping them on ice during the time of the experiment (De Koning et al. 2000). ...
Trypanosoma brucei is an extracellular protozoan parasite that causes human African
trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei
depends on exogenous inorganic phosphate (Pi), but little is known about the transport of Pi
in this organism. In the present study, we showed that the transport of 32Pi across the plasma
membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher
activity at acidic pH. Bloodstream forms presented lower Pi transport in comparison to
procyclic forms, that displayed an apparent K0.5 = 0.093 ± 0.008 mM. Additionally, FCCP (H+-
ionophore), valinomycin (K+-ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited
the Pi transport. Gene Tb11.02.3020, previously described to encode the parasite H+:myo
inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H+:Pi
cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae
and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced
TbHMIT gene expression, compromised cell growth and decreased Pi transport by half. In
addition, Pi transport was inhibited when parasites were incubated in the presence of
concentrations of myo-inositol that are above 300 μM. However, when expressed in Xenopus
laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological
evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may
be only marginally affected by the presence of Pi. These results confirmed the presence of a
Pi carrier in T. brucei, similar to the H+-dependent inorganic phosphate system described in
S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition
of Pi and may be involved in the growth and survival of procyclic forms. In summary, this
work presents the first description of a Pi transport system in T. brucei.
... After washing the cells three times with the same ice-cold solution, they were disrupted using 0.1% sodium dodecyl sulfate (SDS), and the suspension was counted in a scintillation counter (Packard). To obtain blank values, the cells were kept on ice for 60 min in the cold 32 P i -reaction buffer (De Koning et al., 2000). In order to determine the transport affinity and maximal rate, 32 P i uptake assays were carried out at P i concentrations in the range of 1e100 mM in the absence or presence of Na þ . ...
Inorganic phosphate (Pi) is an essential nutrient for all organisms because it is required for a variety of biochemical processes, such as signal transduction and the synthesis of phosphate-containing biomolecules. Assays of ³²Pi uptake performed in the absence or in the presence of Na⁺ indicated the existence of a Na⁺-dependent and a Na⁺-independent Pi transporter in Phytomonas serpens. Phylogenetic analysis of two hypothetical protein sequences of Phytomonas (EM1) showed similarities to the high-affinity Pi transporters of Saccharomyces cerevisiae: Pho84, a Na⁺-independent Pi transporter, and Pho89, a Na⁺-dependent Pi transporter. Plasma membrane depolarization by FCCP, an H⁺ ionophore, strongly decreased Pi uptake via both Na⁺-independent and Na⁺-dependent carriers, indicating that a membrane potential is essential for Pi influx. In addition, the furosemide-sensitive Na⁺-pump activity in the cells grown in low Pi conditions was found to be higher than the activity detected in the plasma membrane of cells cultivated at high Pi concentration, suggesting that the up-regulation of the Na⁺-ATPase pump could be related to the increase of Pi uptake by the Pho89p Na⁺:Pi symporter. Here we characterize for the first time two inorganic phosphate transporters powered by Na⁺ and H⁺ gradients and activated by low Pi availability in the phytopathogen P. serpens.
... It is, for example, common that in vitro cultivated parasites lose almost all of their P2 nucleoside transporter activity, which transports adenine, adenosine and deoxyadenosine (32); apparently the transporter is downregulated under standard culture conditions. Although this does not eliminate all deoxyadenosine uptake, which can also occur by the P1 nucleoside transporter (9), the fact that T. brucei transporters can be differentially expressed under different conditions (33) suggests that in vitro IC50 values may not necessarily reflect in vivo sensitivities. In the human host, the concentration of deoxyadenosine is usually below the detection limit in the blood due to the action of adenosine deaminase. ...
Trypanosoma brucei causes African sleeping sickness, for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mM deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP-knockdown cells. Recombinant TbMTAP exhibited higher turnover number (kcat) and Km values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. In order for deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors.
... A gain in enthalpy with increasing polar surface area of inhibitors suggests that the binding is favored by electrostatic or polar interactions between the ligand and the transporter. Pharmacophore modeling and computational analysis of hENT1 has also indicated the presence of highly positively charged residues in the ligand binding pocket which interact with the negatively charges moieties of NBMPR [35]. Furthermore, the manifestation of a single binding mode (enthalpy driven) by three chemically distinct inhibitors and adenosine may suggest that the binding pocket of hENT1 will not tolerate diversity in the types of interactions that result in high affinity binding. ...
... Trypanosomatids are protozoan parasites that cannot synthesize purines de novo [20,38,45]. It has been postulated that these ecto-nucleoside triphosphate diphosphohydrolases could play a role in the salvage of purines from the host in Leishmania amazonensis [18,20], Trypanosoma cruzi [40] and Trypanosoma brucei [38]. ...
Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), also known as ecto-ATPases and/or ecto- apyrases, are integral membrane glycoproteins or soluble enzymes that are dependent on divalent cations. These ecto- enzymes are important ecto-nucleotidases that are characterized by the ability to hydrolyze nucleoside triphosphates and nucleoside diphosphates to the monophosphate form. The hydrolysis of nucleoside monophosphates to nucleosides such as adenosine may then be catalyzed by the action of ecto-5´nucleotidases. The present study reviews the sequential hy- drolysis of ATPADPAMPadenosine catalyzed by these ecto-enzymes from different trypanosomatids. These reactions participate in the salvage of purines in these parasites and simultaneously interfere with the establishment of in- fection and changes in the host immune response.
... Since trypanosomes are unable to synthesize purines de novo (35), it is conceivable that they need to stimulate the transport of nucleobases when purines become scarce as cell density increases. Indeed, it has been shown that trypanosomes and Leishmania respond to purine starvation by increasing their transport capacity (36,37). Interestingly, we found that the addition of guanosine to late log cultures reversed the accumulation of NT8 mRNA and increased the abundance of an otherwise downregulated mRNA. ...
We have compared the transcriptomes of cultured procyclic Trypanosoma brucei cells in early and late logarithmic phases and found that ∼200 mRNAs were differentially regulated. In late log phase cells,
the most upregulated mRNA encoded the nucleobase transporter NT8. The 3′ untranslated region (UTR) of NT8 contains a short stem–loop cis-element that is necessary for the regulation of NT8 expression in response to external purine levels. When placed in the 3′-UTR of an unregulated transcript, the cis-element is sufficient to confer regulation in response to purines. To our knowledge, this is the first example of a discrete
RNA element that can autonomously regulate gene expression in trypanosomes in response to an external factor and reveals an
unprecedented purine-dependent signaling pathway that controls gene expression in eukaryotes.
... However, the regulation of this pathway in response to changes in the extracellular purine milieu is poorly understood. Earlier studies in Leishmania and related parasites have revealed a marked augmentation in cell surface activities corresponding to 39nucleotidase/nuclease (39NT/NU) and membrane-bound acid phosphatase in response to purine starvation [13,[28][29][30][31][32], and studies from our own laboratory [8,33], as well as others [12,34,35], have shown augmentation of nucleoside and nucleobase transport activities and proteins. Purine starvation is easily induced in vitro by the withdrawal of purines from the growth medium, and we have previously developed conditions where the response to purine stress is both robust and readily tractable [8]. ...
The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.
... Following three washes with the same ice-cold buffer, the cells were disrupted by adding 0.1% SDS and the radioactivity was measured using a scintillation counter. To obtain the blank uptake values, cells were exposed to a cold 32 P i reaction mixture and then kept on ice for 60 min [21]. To determine the effect of pH on P i uptake, the same buffer described above was used, with the addition of 10 mM HEPES/MES/Tris at various pH values. ...
... This coincides with the evolution of 11 MSP-C genes in T. vivax, a gene that is single-copy in all other species (see Fam46 CSP page, andTable S1). The surface functional repertoire also diverges through gene gain, for example among Fam61 genes (nucleoside/nucleobase transporters), required to scavenge host purines and are functionally differentiated with respect to both parasite life stage and substrate58596061. The Fam61 phylogeny shows that multiple gene duplications have occurred in both T. brucei and T. congolense (see Fam61 CSP page). ...
The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome.
... Following 3 washes with the same ice-cold buffer, the cells were disrupted by addition of 0.1% SDS and the suspension counted in a scintillation counter (Packard). Blank uptake values were obtained by exposing the cells to a cold 32 P i -reaction mixture kept on ice for 60 min [18]. For determination of the transport affinity (K 0.5 ) and maximal rate (V max ), 32 P i uptake was measured at P i concentrations ranging 5-500 μM in the absence or presence of Na + . ...
Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake.
We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase.
T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation.
Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli.
This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.
... More recently, it was also shown that resistance to eflornithine is linked to the loss of a single amino acid transporter gene, TbAAT6 (43). The close correlations between drug efficacy and transport efficiency, as well as between drug resistance and changes in transporter function or expression (10, 17, 22, 30, 42 ), reinforce the importance of studying drug-transporter interactions, especially in cases of parasitic disease. In addition to TbAT1 and P2, two additional drug transporters have been described for pentamidine and melaminophenyl arsenicals in T. brucei, HAPT1 and LAPT1, explaining the absence of high levels of resistance for these compounds in tbat1 / (7, 34). ...
Current therapies for human African trypanosomiasis (HAT) are unsatisfactory and under threat from emerging drug resistance linked to the loss of transporters, e.g., the P2 aminopurine transporter (TbAT1). Here we compare the uptake and trypanocidal properties of furamidine (DB75), recently evaluated in clinical trials against stage 1 (haemolymphatic) HAT, and two aza analogues, DB820 and CPD0801 (DB829), which are candidate compounds for treatment of stage 2 (neurological) disease. Values of 50% inhibitory concentrations (IC50s) determined in vitro against both wild-type and transporter mutant parasites were submicromolar, with DB75 trypanotoxicity shown to be better than and DB820 trypanotoxicity similar to that of the widely used veterinary trypanocide diminazene, while CPD0801 was less active. Activity correlated with uptake and with the minimum drug exposure time necessary to kill trypanosomes: DB75 accumulated at double and 10-fold the rates of DB820 and CPD0801, respectively. All three compounds inhibited P2-mediated adenosine transport with similar Ki values, indicating affinity values for this permease in the low to submicromolar range. Uptake of DB75, DB820, and CPD0801 was significantly reduced in tbat1-/- parasites and was sensitive to inhibition by adenine, showing that all three compounds are substrates for the P2 transporter. Uptake in vitro was significantly less than that seen with parasites freshly isolated from infected rats, correlating with a downregulation of P2 activity in vitro. We conclude that DB75, DB820, and CPD0801 are actively accumulated by Trypanosoma brucei brucei, with P2 as the main transport route. The aza analogues of DB75 accumulate more slowly than furamidine itself and reveal less trypanocidal activity in standard in vitro drug sensitivity assays.
... Similar to other protozoan such as Leishmania species (Meyer-Fernandes et al., 1997;Berrêdo-Pinho et al., 2001; 0304-4017/$ -see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.01.061 de Almeida Marques-da-Silva et al., 2008), including Trypanosoma cruzi (Bisaggio et al., 2003;Meyer-Fernandes et al., 2004), Trypanosoma brucei (de Koning et al., 2000;Leite et al., 2007), Giardia duodenalis (Pinheiro et al., 2008) and Trichomonas vaginalis (Tasca et al., 2003), T. foetus is devoid of a de novo synthesis pathway for purine nucleotides (Wang et al., 1983), depending instead on salvaging purines from the host environment. In addition to their nutritional importance, extracellular nucleotides may participate in signaling functions through purine receptors located on the cell membrane. ...
Tritrichomonas foetus is the causative agent of sexually transmitted trichomoniasis in cattle. In females, the infection can be associated with infertility, vaginitis, endometritis, abortion or pyometra, leading to significant economic losses in cattle raising. T. foetus is devoid of the ability to synthesize purine nucleotides de novo, depending instead on salvaging purines from the host environment. Ecto-5'-nucleotidase catalyzes the final step of extracellular nucleotide degradation, the hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and Pi. In this work we show that living, intact cells of T. foetus were able to hydrolyze 5'AMP at a rate of 12.57 ± 1.23 nmol Pi × h(-1) × 10(-7) cells at pH 7.2 and the 5'AMP hydrolysis is due to a plasma membrane-bound ecto-enzyme activity. The apparent K(m) for 5'AMP was 0.49 ± 0.06 mM. In addition to 5'AMP, the enzyme hydrolyzed all substrate monophosphates tested except 3'AMP. No divalent metals or metal chelators were able to modulate enzyme activity. Phosphatase inhibitors did not have an effect on ecto-5'-nucleotidase activity while ammonium molybdate did inhibit the activity in a dose dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. These results indicate the existence of an ecto-5'-nucleotidase that may play a role in the salvage of purines.
... Trypanosomatid protozoans are incapable of synthesising purines de novo and must rely on their hosts for these essential nutrients (Hassan and Coombs, 1988;De Koning et al., 2000;Berrêdo-Pinho et al., 2001;Meyer-Fernandes, 2002;Meyer-Fernandes et al., 2004;Pinheiro et al., 2006;Leite et al., 2007). Leishmania species have membrane bound 3 0 -nucleotidases on their extracellular surface that hydrolyse extracellular 3 0 -nucleotides or nucleic acids to form nucleosides (Dwyer and Gottlieb, 1984;Gbenle, 1993;Lakhal-Naouar et al., 2008). ...
In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3'AMP 10 times more than 5'AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3'nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3'nucleotidase activity was not observed on ecto-5'nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3'nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage-parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3'nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite-macrophage interaction.
... Related work by Gero and colleagues (Hall et al., 1996; Hall et al., 1998) confirmed this upregulation in C. luciliae, and experiments employing Xenopus oocytes suggested that the enhancement of transport activity upon purine starvation was not accompanied by a corresponding increase in mRNAs encoding these transporters. Additional studies extended upregulation by purine starvation to 3'-nucleotidase in L. donovani (Sacci et al., 1990) and to purine transporters in Trypanosoma brucei (de Koning et al., 2000). Hence, both transport and salvage enzymes appear to be regulated by extracellular purine levels in multiple kinetoplastid protozoa to maximize import of these nutrients under limiting conditions. ...
Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations.
... Wong and M.P. Barrett, unpublished observation). Regulation of transport capacity in response to growth stage or substrate availability appears to be a general feature for transporters in kinetoplastidae [19,20] and we propose that the variation in quantity of CPD0801 transported could be a function of parasitaemia at the time of har- vest. The applicability of our assay to other fluorescent compounds was tested by measuring uptake of the phenanthridine drug ethidium by the same trypanosome strain, commonly used as a veterinary trypanocide in sub-Saharan Africa under the trade name Homidium [15]. ...
Drug therapies currently used for second stage Human African Trypanosomiasis (HAT) exhibit problems with toxicity, difficulty of administration, and resistance linked to the loss of transporter function. Key to the development of new drugs for HAT is a better understanding of the transport properties of candidate compounds. Standard methods for studying transport utilize radio-labelled permeant or HPLC-MS, however the natural fluorescence of many trypanocidal compounds can be exploited. Here we present a fluorescence-based assay for measuring uptake, by trypanosomes, of CPD0801, a drug candidate for second stage HAT. Sample fluorescence is measured in a 96-well format using a benchtop fluorimeter. Our method is directly applicable to the study of other diamidines with similar fluorescent properties and readily adapted for use with other cell types or fluorescent molecules as we demonstrate for the veterinary trypanocide ethidium.
... It is well known that some protozoa parasites are deficient in de novo synthesis of purine nucleotides. Besides G. duodenalis (Pinheiro et al., 2008), other parasites like Trypanosoma cruzi (Bisaggio et al., 2003;Meyer-Fernandes et al., 2004), Trypanosoma brucei (de Koning et al., 2000;Leite et al., 2007), Leishmania spp. (Meyer-Fernandes et al., 1997;Berrêdo-Pinho et al., 2001) and trichomonad species (Jesus et al., 2002;Tasca et al., 2003) depend on the salvage of purines from the host environment. ...
In this work, we biochemically characterized the ecto-5'-nucleotidase activity present on the surface of the living trophozoites of Giardia duodenalis. Two sequences of the 5'-nucleotidase family protein were identified in the Giardia genome. Anti-mouse CD73 showed a high reaction with the cell surface of parasites. At pH 7.2, intact cells were able to hydrolyze 5'-AMP at a rate of 10.66 ± 0.92 nmol Pi/h/10(7) cells. AMP is the best substrate for this enzyme, and the optimum pH lies in the acidic range. No divalent cations had an effect on the ecto-5'-nucleotidase activity, and the same was seen for NaF, an acid phosphatase inhibitor. Ammonium molybdate, a potent inhibitor of nucleotidases, inhibited the enzyme activity in a dose-dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. The results indicate the existence of an ecto-5'-nucleotidase that could play a role in the salvage of purines.
... In view of the above results, as well as of previous studies with diamidine-resistant trypanosomes (see Introduction), it was deemed very likely that ASPT1 is identical to the P2 aminopurine transporter. One prediction from this hypothesis was that ASPT1 would not be present in procyclic forms of T. brucei brucei, since the P2 transporter is not expressed in this life cycle stage (De Koning et al., , 2000b. This prediction was borne out with the observation that transport of 25 nM, 250 nM, or 1 M [ 3 H]pentamidine by procyclic T. brucei brucei was not inhibited by up to 1 mM adenosine or adenine (not shown). ...
The trypanocidal action of pentamidine is dependent on the rapid, selective accumulation of this drug by the parasite. We have investigated pentamidine transport by the bloodstream and procyclic life cycle stages of Trypanosoma brucei brucei. In bloodstream forms, 50 to 70% of [(3)H]pentamidine was transported by an adenosine-sensitive pentamidine transporter (ASPT1) that displayed a K(m) value of 0.26 +/- 0.03 microM and K(i) values of 0.45 +/- 0.04 and 2.5 +/- 0.8 microM for adenine and berenil, respectively. These values are very similar to those for inhibition of [(3)H]adenosine uptake by the P2 adenosine/adenine transporter, suggesting that ASPT1 and P2 may be identical. The remaining 30 to 50% of [(3)H]pentamidine transport was mediated by a low-capacity high-affinity pentamidine transporter (HAPT1) and a high-capacity low-affinity pentamidine transporter (LAPT1), with K(m) values of 36 +/- 6 nM and 56 +/- 8 microM, respectively. HAPT1 was inhibited by propamidine but displayed only low affinity to berenil and stilbamidine, whereas LAPT1 was not inhibited by any of these diamidines. Neither transporter was inhibited by melarsen oxide. In procyclics, an HAPT1-analog (procyclic pentamidine transporter; PPT1) was characterized, but no adenosine-sensitive pentamidine transport could be detected. Treatment with ionophores revealed that PPT1 may be a proton/pentamidine cotransporter.
... It has been shown that purine salvage is regulated by extracellular purine concentrations (Gero et al., 1997;De Koning et al., 2000). Since the in vitro-cultivated and the blood stream-derived trypanosomes are exposed to different levels of purine, it may be argued that this could have some influence on the measurable transporter activity. ...
Resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake. The transport of melaminophenyl arsenicals into Trypanosoma brucei was shown to be mediated by an unusual adenosine nucleoside transporter, P2 (Carter and Fairlamb, 1993), and the loss of this transporter is associated with resistance to melaminophenyl arsenicals in these parasites. To further understand the mechanisms of arsenical resistance, we generated several lines of Mel Cy-resistant T. evansi from a drug-sensitive isolate using both in vivo and in vitro selection methods. Uptake of the melaminophenyl arsenical, Mel Cy on the P2 transporter was studied in the drug-sensitive as well as Mel Cy-resistant parasites, by means of inhibition of Mel Cy-induced lysis of trypanosomes, in an in vitro lysis assay. Adenosine uptake was also investigated using competition inhibition assays. Our study shows that T. evansi, TREU 1840, possesses the P1/P2 adenosine transport system as reported in T. brucei and T. equiperdum. However, in T. evansi, the P2 transporter is the larger transport process instead of the P1. The P2 transporter in T. evansi mediated the uptake of Mel Cy in the drug-sensitive parasites. The P2 was retained in all the arsenical-resistant T. evansi lines studied. However, the activity of the transporter was reduced to different extents in the different-resistant lines. The residual P2 activity related well to the levels of drug resistance in each line, suggesting that P2 activity could be an important marker for arsenical resistance. Furthermore, important differences were observed between the in vivo- and the in vitro-selected arsenical-resistant parasites suggesting that there may be differences in resistance phenotypes selected on the field.
... The complexity of purine metabolism makes it difficult to study, especially in intracellular parasites. Metabolic studies on Trypanosomatids evidenced that purine stress induced adaptations in its metabolism [11,12]. Apicomplexa have been less studied, essentially Toxoplasma gondii [13,14], Plasmodium falciparum [15], Babesia [16] and recently, HGXPRT was evidenced in Cryptosporidium parvum [17]. ...
The effect of purine nucleosides on the in vitro growth of Cryptosporidium parvum was studied. Culturing the parasite in THP-1 cells for 72 h in growth medium supplemented with adenosine or inosine improved the parasite yields especially in the first 48 h. Similar results were obtained with parasites cultured in Madin-Darby bovine kidney cells and incubated for 24 h with inosine. The addition of inosine to 72-h cultures enhanced the growth of C. parvum in THP-1 cells, especially the trophic stages, whereas the analogue formycin B was toxic to the parasites and induced a marked decrease in the gamont stages. The monitoring of the added purine nucleosides by high performance liquid chromatography showed that at 37 degrees C in the presence of THP-1 cells, a rapid uptake of inosine occurred with hypoxanthine being the main purine present after 2 h in the medium.
... In contrast, the value for V max at the P2 in the drug-resistant trypanosomes was decreased 10-fold. In related studies on purine transport in other trypanosomatids (de Koning et al. 2000), observed increased V max of adenosine in Crithidia luciliae, which was abolished by treatment with cyclohexidine (inhibits protein synthesis). These authors suggested that increased V max was due to increased numbers of transporters. ...
Studies of the kinetics of adenosine transport were carried out on the P1 and P2 transporters of drug-sensitive Trypanosoma evansi and its cloned derivative, resistant to the melaminophenyl arsenical Mel CY. Characterization of adenosine uptake was made by estimation of the maximum concentration taken up at time infinity (Amax). Amax on the P2 transporter of sensitive T. evansi was greater than Amax on the P1 transporter. Amax of the P2 transporter was significantly decreased in drug-resistant trypanosomes. The effect of adenosine concentration and inhibitors, on the rate of adenosine uptake, was described by Michaelis-Menten equations. In sensitive T. evansi, the maximum velocity of adenosine uptake (Vmax) of the P2 transporter was 2-fold greater than Vmax of the P1 transporter. The Vmax of the P2 transporter in resistant parasites was reduced 9-fold. The binding constants Km and Ki on the P2 transporter of resistant T. evansi, showed that resistance was associated with an increased affinity for adenosine, and a decreased affinity for adenine and Berenil. We suggest that resistance to melaminophenyl arsenicals in T. evansi, occurs via 2 mechanisms: (1) a reduction in the number of expressed P2 transporter molecules resulting in decreased uptake of melaminophenyl arsenicals; (2) a change in the binding properties of the P2 transporter.
Giardia duodenalis is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as giardiasis. Similar to other parasites, G. duodenalis must take advantage of environmental resources to survive, such as inorganic phosphate (Pi) availability. Pi is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H⁺:Pi-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, Pi transport was linear for up to 15 min, and the cell density was 3 × 10⁷ cells/ml. Physiological variations in pH (6.4 to 8.0) did not influence Pi uptake. This Pi transporter had a high affinity, with K0.5 = 67.7 ± 7.1 µM Pi. SCH28080 (inhibitor of H⁺, K⁺-ATPase), bafilomycin A1 (inhibitor of vacuolar H⁺-ATPase), and FCCP (H⁺ ionophore) were able to inhibit Pi transport, indicating that an H⁺ gradient in the cell powered uphill Pi movement. PAA, an H⁺-dependent Pi transport inhibitor, reduced cell proliferation, Pi transport activity, and GdPHO48 mRNA levels. Pi starvation stimulated membrane potential-sensitive Pi uptake coupled to H⁺ fluxes, increased GdPho84 expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize Pi as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized Pi can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits Pi influx. Together, these results reinforce the hypothesis that Pi is a crucial nutrient for G. duodenalis energy metabolism.
Schistosomes are responsible for the parasitic disease schistosomiasis, an acute and chronic parasitic ailment that affects more than 240 million people in 70 countries worldwide. It is the second most devastating parasitic disease after malaria. At least 200,000 deaths per year are associated with the disease. In the absence of the availability of vaccines, chemotherapy is the main stay for combating schistosomiasis. The antischistosomal arsenal is currently limited to a single drug, Praziquantel, which is quite effective with a single-day treatment and virtually no host-toxicity. Recently, however, the question of reduced activity of Praziquantel has been raised. Therefore, the search for alternative antischistosomal drugs merits the study of new approaches of chemotherapy.
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as (13C)GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as (13C)ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction.
Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (P(i)) but little is known about energy metabolism and transport of P(i) across the plasma membrane in Leishmania sp.
We investigated the kinetics of 32P(i) transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:P(i) and H+:P(i) cotransporters.
The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K(+)-ATPase) all inhibited the transport of P(i). This transport showed Michaelis-Menten kinetics with K0.5 and V(max) values of 0.016 +/- 0.002 mM and 564.9 +/- 18.06 pmol x h(-1) x 10(-7) cells, respectively. These values classify the P(i) transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, P(i) transport and LiPho84 gene expression were modulated by environmental P(i) variations.
These findings confirm the presence of a P(i) transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum.
This work provides the first description of a PHO84-like P(i) transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.
There is a gulf between the enormous information content of the various genome projects and the understanding of the life of the parasite in the host. In vitro studies with adult Schistosoma mansoni using several substrates suggest that the excretory system contains both P-glycoproteins and multiresistance proteins. If both these families of protein were active in vivo, they could regulate parasite metabolism and be responsible for the excretion of drugs. During skin penetration, membrane-impermeant molecules of a wide range of molecular weights can be taken into the cercaria and schistosomulum through the nephridiopore, through the surface membrane or through both. We speculate that this uptake process might stimulate novel signalling pathways involved in growth and development.
The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.
Prodrug design strategies have been employed to improve the delivery of drugs with undesirable pharmacokinetic properties such as chemical stability and lack of specificity. Targeted prodrug design represents a new strategy for site-directed and efficient drug delivery. Targeting of drugs to transporters and receptors to aid in site-specific carrier-mediated absorption is emerging as a novel and clinically significant approach. Various prodrugs have been successful in achieving the goals of enhanced bioavailability and are, therefore, considered to be an important tool in biopharmaceutics. This review highlights the advances in prodrug design targeted towards membrane transporters/receptors in the past few years.
While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from
many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters
of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both
the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity
transport activity for adenine and hypoxanthine, with Km values of 2.1 ± 0.6 and 0.66 ± 0.22 μm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine.
A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by ∼80% and H4 transport
activity by ∼90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals
to be identified at the molecular level.
Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol,
the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of
P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and
melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine,
while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity
might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected
with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were
still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl
arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.
Protozoan parasites are incapable of synthesizing purine nucleotides de novo and so must salvage preformed purines from their hosts. This process of purine acquisition is initiated by the translocation of preformed host purines across parasite or host membranes. Here, we report upon the identification and isolation of DNAs encoding parasite nucleoside transporters and the functional characterization of these proteins in various expression systems. These potential approaches provide a powerful approach for a thorough molecular and biochemical dissection of nucleoside transport in protozoan parasites.
Allopurinol is a hypoxanthine analogue used to treat Leishmania infections that also displays activity against the related parasite Trypanosoma brucei. We have investigated the ease by which resistance to this drug is established in Trypanosoma brucei brucei and correlated this to the mechanisms by which it is accumulated by the parasite. Long-term exposure of procyclic T. b. brucei to 3mM allopurinol did not induce resistance. This appears to be related to the fact that allopurinol was taken up through two distinct nucleobase transporters, H1 and H4, both with high affinity for the drug. The apparent Km for [3H]allopurinol transport by H4 (2.1+/-0.4 microM) was determined by expressing the encoding gene in Saccharomyces cerevisiae. Long-term allopurinol exposure did not change Km (hypoxanthine), Ki (allopurinol), or Vmax values of either H1 or H4 transporters and the cells retained their ability to proliferate with hypoxanthine as sole purine source. This study shows that transport-related resistance to purine antimetabolites is not easily induced in Trypanosoma spp. as long as uptake is mediated by multiple transporters.
To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.
Adenosine transport at 22 degrees C in procyclic forms of Trypanosoma brucei brucei was investigated using an oil-inhibitor stop procedure for determining initial rates of adenosine uptake in suspended cells. Adenosine influx was mediated by a single high affinity transporter (Km 0.26 +/- 0.02 microM, Vmax 0.63 +/- 0.18 pmol/10(7) cells s-1). Purine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (Ki 0.18-5.2 microM). Purine nucleobases and pyrimidine nucleosides and nucleobases had no effect on adenosine transport. This specificity of the transporter appears to be similar to the previously described P1 adenosine transporter in bloodstream forms of trypanosomes. Uptake of adenosine was Na+-independent, but ionophores reducing the membrane potential and/or the transmembrane proton gradient (monitored with the fluorescent probes bis-(1,3-diethylthiobarbituric acid)-trimethine oxonol and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, respectively) inhibited adenosine transport. Similarly, an increase in extracellular pH from 7.3 to 8.0 reduced adenosine influx by 30%. A linear correlation was demonstrated between the rate of adenosine transport and the protonmotive force. Adenosine uptake was accompanied by a proton influx in base-loaded cells and was also shown to be electrogenic. These combined results indicate that transport of adenosine in T. brucei brucei procyclics is protonmotive force-driven and strongly suggest that the adenosine transporter functions as an H+ symporter.
Promastigotes of the protozoan parasite Leishmania major exhibit high affinity uptake of folate (Kt = 0.7 microM) and methotrexate (MTX) (Kt = 1.8 microM) which is saturable and sensitive to metabolic poisons. Influx of folate and MTX is competitively inhibited by 5-formyltetrahydrofolate and p-aminobenzoic acid-glutamate, but not by 4-deoxy-4-amino-10-methylpteroate, biopterin, or pteroate. A single carrier is inferred for both folate and MTX transport, as the Ki of each inhibitor for both folate and MTX influx is the same, and the apparent affinities (Kt) of the substrates folate and MTX are identical to their respective Ki values for inhibition of MTX and folate uptake. Folate influx is specifically regulated according to cellular growth phase, as stationary phase cells exhibit 7% of the Vmax of log phase cells, while energy-dependent glucose uptake is only moderately reduced in stationary phase. Folate influx is also regulated by external folate levels, as cells grown in 5 microM folate exhibit 30% of the Vmax of cells grown in folate-depleted medium. Comparison of bacterial, mammalian, and Leishmania folate transport activities indicates considerable diversity in both biochemical and regulatory properties, and suggests the possibility that selective inhibition or manipulation of folate transport may be exploited in parasite chemotherapy.
Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.
The African trypanosome, Trypanosoma brucei brucei, possesses at least two nucleoside transporter systems designated P1 and P2, the latter being implicated in the selective
uptake of melaminophenyl arsenical drugs. Since arsenical-resistant trypanosomes show cross-resistance in vivo to aromatic diamidines, we have investigated whether these drugs are also substrates for the P2 nucleoside transporter. In
melarsen-sensitive T. b. brucei, the diamidines, including the commonly used trypanocides, pentamidine and berenil, were found to abrogate lysis induced
by the P2 transport of melarsen oxide in vitro. Measurement of [ring-H]pentamidine transport in melarsen-sensitive T. b. brucei, demonstrated that uptake is carrier-mediated, with a K of 0.84 μM and a V of 9.35 pmol s (10 cells). Pentamidine transport appears to be P2-mediated in these cells, as pentamidine strongly inhibited uptake of [2′,5′,8-H]adenosine by the P2 transporter, with a K of 0.56 μM. Furthermore, [ring-H]pentamidine transport was blocked by a number of P2 transporter substrates and inhibitors, as well as by other diamidine
drugs. Analysis of the uptake of pentamidine and other diamidines in melarsen-resistant trypanosomes in vitro and in vivo, which also show differential levels of resistance to these compounds in vivo, indicated that P2 transport was altered in these cells and that accumulation of these drugs was markedly reduced.
The melaminophenyl arsenical melarsoprol is still used to treat African sleeping sickness, a disease caused by parasitic protozoa of the Trypanosoma brucei subgroup. Based on the observation that melamine antagonizes the trypanocidal activity of this class of drugs, we investigated whether other physiological compounds could compete for the same receptor. Here we report that the in vitro trypanolytic effect of melarsen oxide can be specifically abrogated by adenine, adenosine and dipyridamole, all of which compete for uptake by an adenosine transporter. Melarsen-sensitive trypanosomes have two high-affinity adenosine transport systems: a P1 type, which also transports inosine; and a P2 type, which also transports adenine and the melaminophenyl arsenicals. Melarsen-resistant trypanosomes lack P2 adenosine transport, suggesting that resistance to these arsenicals is due to loss of uptake.
Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine K(m) 2.0 microM, V(max) 3.3 nmol/min per 10(8) cells; cadaverine K(m) 13.4 microM, V(max) 3.9 nmol/min per 10(8) cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027].
The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.
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The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22°C was rapid and saturable, exhibiting an apparent Km, of 9.3±2.0 M and a Vmax of 4.5±0.8 pmol · (107cells)−1· s−1. All the natural purine nucleobases tested (Km 1.8–7.2 M), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N, N′-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2′, 7′-bis-(carboxyethyl)-5, 6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi, the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electro-genic. These data show that hypoxanthine uptake in T b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.
Hypoxanthine transport in the insect trypanosome, Crithidia
luciliae, was activated in purine depleted conditions. The existence
of 2 hypoxanthine transport mechanisms was established. The first, a non-saturable
diffusion system, present in purine replete conditions, exhibited properties
that were different from the second transport system which was evident
only during purine depleted conditions (purine stress). The rate of transport
under purine stress was elevated approximately 8-fold over that in replete
conditions. This transporter was saturable with a Km
of 3·9 μM for hypoxanthine. The transporter substrate specificity
included other purine bases, e.g. adenine and guanine, and the purine nucleoside,
adenosine. These inhibited hypoxanthine transport competitively with Ki
values of 2 μM, 3 μM and 42 μM respectively.
Coincident with the increase of hypoxanthine transport under purine stress,
the transport of adenosine increased 4-fold and the activity of the 3′-nucleotidase
ectoenzyme also increased significantly. Under purine stress the concurrent
increase of hypoxanthine and adenosine transport and the increase in 3′-nucleotidase
activity could be repressed with either the supplementation of excess purines
or by cycloheximide. This study of purine salvage mechanisms in Crithidia
luciliae illustrates the successful adaptation of the parasite to
nutritional insufficiency.
The mechanism and inhibitor sensitivity of hypoxanthine transport by bloodstream forms of Trypanosoma brucei brucei was investigated. The dose response curve for the inhibition of hypoxanthine transport (1 μM) by guanosine was biphasic; ≈90% of transport activity was inhibited with a Ki value of 10.8±1.8 μM, but 10% of the activity remained insensitive to concentrations as high as 2 mM. These two components of hypoxanthine transport are defined as guanosine-sensitive (H2) and guanosine-insensitive (H3). Hypoxanthine influx by both components was saturable, but there was a marked difference in their Km values (123±15 nM and 4.7±0.9 μM for H2 and H3, respectively) although the Vmax values (1.1±0.2 and 1.1±0.1 pmol (107 cells)−1 s−1, n=3) were similar. Hypoxanthine uptake via the H2 carrier was inhibited by purine bases and analogues as well as by some pyrimidine bases and one nucleoside (guanosine), whereas the H3 transporter was sensitive only to inhibition by purine nucleobases. H2-mediated hypoxanthine uptake was inhibited by ionophores, ion exchangers and the potential H+-ATPase inhibitors, N,N′-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM). Measurements of the intracellular pH and membrane potential of bloodstream trypanosomes in the presence and absence of these agents established a linear correlation between protonmotive force and rate of [3H]hypoxanthine (30 nM) uptake. We conclude that hypoxanthine transport in bloodstream forms of T. b. brucei occurs by two transport systems with different affinities and substrate specificities, one of which, H2, appears to function as a H+/hypoxanthine symporter.
It has been shown previously that starvation of the trypanosomatid protozoan Crithidia luciliae for purines and/or inorganic phosphate results in increased levels of a surface membrane-associated 3'-nucleotidase/nuclease (3'-N'ase) activity which hydrolyzes both 3'-ribonucleotides and nucleic acids, thereby permitting the organisms to transport these essential nutrients across their cell membranes. A polypeptide with the requisite catalytic properties has been identified by an in situ gel activity assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In current studies, differential synthesis of the protein responsible for the 3'-N'ase activity was not demonstrable by comparisons of SDS-PAGE patterns of nutrient-replete or purine-starved parasites metabolically labeled with either [35S]methionine, [3H]leucine, or [3H]tyrosine. However, surface labeling of nutrient-replete and purine-starved cells revealed the enhanced expression of an 125I surface-labeled 43-kDa protein which comigrated with the 3'-N'ase activity in one- and two-dimensional electrophoretic systems. The amount of this surface-labeled peptide correlated with the level of 3'-N'ase activity as measured by test tube assay. Refeeding adenosine to purine-starved cells led to the loss of both the enzyme activity and the surface iodinatable 43-kDa band as a result of renewed cell division. Starvation of these organisms for phosphate also led to the enhanced expression of the 43-kDa radioiodinatable band. The results indicated that the 3'-N'ase protein, itself, is differentially expressed at the cell surface under conditions which lead to increased enzyme activity.
Crithidia fasciculata, a nonpathogenic relative of the leishmanial and trypanosomal pathogens of humans and animals, showed a 3'-ribonucleotidase activity similar to that in Leishmania donovani. The level of 3'-nucleotidase activity in Crithidia was regulated by the availability of purines in the culture medium. Specifically, organisms obtained from culture medium depleted of purines contained elevated levels of enzyme activity compared to those grown in complete medium. The 3'-nucleotidase, located at the cell surface, may serve as a first step in purine salvage for these protozoa, which are unable to synthesize the purine ring de novo.
Nucleoside salvage pathways are vital to the parasitic protozoan Trypanosoma cruzi, and have become important targets in the development of new chemotherapeutic agents against this organism. We produced a mutant T. cruzi clone with a defect in the uptake of the adenosine analogue tubercidin which allowed us to hypothesize that there are at least two distinct nucleoside transport pathways in this parasite. The mutant shows a marked defect in the uptake of tubercidin and thymidine, whereas the uptake of adenosine and inosine are normal. Inhibition and metabolic studies suggest that the defect is related to transport and that there are two transport processes relatively specific for purines and pyrimidines, respectively, although tubercidin is transported via the latter. This is similar to the reported dual nucleoside transport pathways in Leishmania donovani and may be a common system in the Trypanosomatidae. These transport processes are markedly different from those which have been described for mammalian cells and may play an important role in the design of strategies for the chemotherapy of human infection with these pathogenic parasites.
A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.
The pathways leading to purine and pyrimidine nucleotide production in members of the family Trypanosomatidae are discussed with special emphasis on data relating to pathogenic species published from 1974 to 1983 inclusive. Trypanosomes and leishmania in general lack a de novo purine biosynthetic pathway, but have a multiplicity of possible routes for purine salvage. In contrast, pyrimidine nucleotides can be produced by either de novo or salvage pathways. The properties of these pathways in trypanosomatids are compared and contrasted with those of their hosts.
Nucleoside transport was evaluated in the trypanosomatid Crithidia luciliae by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent Km values for adenosine and guanosine were 9.34 +/- 1.30 and 10.6 +/- 2.60 microM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer, L-adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae.
Following the demonstration that the transport of melaminophenyl arsenical drugs in Trypanosoma brucei is dependent upon an unusual adenosine nucleoside transporter (Carter and Fairlamb, Nature 361 (1993) 173-175) we have investigated adenosine transport in the related parasite Trypanosoma equiperdum (Botat1.1) and a cloned derivative resistant to the diamidine drug berenil (diminazene aceturate) with limited cross-resistance to the melaminophenyl arsenical cymelarsen. The parental strain possesses a bipartite adenosine transport system consisting of one component which is inhibited in a dose-dependent and saturable manner with increasing concentrations of inosine and a second component which is similarly inhibited by adenine. Uptake of adenosine on this second transporter is also inhibited in a dose-dependent fashion by berenil and cymelarsen. Both transporters have high affinity for adenosine (apparent Km values of 0.60 and 0.70 mM and Vmax values of 8.4 and 6.9 pmol (s (10(8) trypanosomes))-1 at 25 degrees C, respectively). Thus T. equiperdum shares with T. brucei a system comprising two adenosine transporters named P1 and P2, respectively. The P1 transporter is similar in the sensitive and resistant T. equiperdum clones, whereas the P2 transporter has reduced transport capacity at physiological adenosine concentration and decreased affinity for adenosine in the drug-resistant clone.
During the growth cycle of the protozoan parasite Crithidia luciliae, there was a dramatic concomitant increase in the rate of adenosine and guanosine transport and 3' nucleotidase (3'NTase) activity after 72-94 hr. The simultaneous increased activities of the nucleoside transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine (100 microM). C. luciliae grown in purine-replete medium (> or = 75 microM adenosine) exhibited low rates of adenosine and guanosine transport whilst parasites transferred to a defined serum-free medium containing < or = 7.5 microM adenosine demonstrated elevated levels of both adenosine and guanosine transport up to 25- to 40-fold. The increased activity of the nucleoside transporters was inhibited by cycloheximide (10 microM). Under conditions of purine depletion 3'AMP and 3'GMP inhibited the adenosine and guanosine transporters, respectively. However, in the presence of a purine supplement (100 microM), neither 3'AMP nor 3'GMP was an effective inhibitor of nucleoside transport. Our results link the increased activity of the nucleoside transporters to the increased activity of the 3'NTase, indicating the activation of a purine salvage system not previously reported in other organisms.
Continuous exposure of Trypanosoma evansi bloodstream forms to Cymelarsan in vitro resulted in the induction of resistance to the drug over a period of 4.5 months. Induction of resistance to Cymelarsan was accompanied by increased resistance to both Arsobal and Berenil, but Cymelarsan-resistant trypanosomes remained sensitive to Suramin and Antrycide. Since resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake, the adenosine metabolism in drug-sensitive and drug-resistant clones was measured. The initial rate of [3H]-adenosine uptake was much higher in sensitive trypanosomes than in drug-resistant parasites, but the amount of radiolabel accumulated by each population was similar. Both adenine and inosine inhibited incorporation of [3H]-adenosine in each population, but in quite different ways. Adenine inhibited more than 80% of adenosine incorporation in drug-sensitive trypanosomes but suppressed less than half of this process in the resistant population. In contrast, inosine inhibited only 10-15% of adenosine incorporation in the sensitive parasites but was inhibitory to a much greater extent in resistant trypanosomes. Lysis of drug-sensitive trypanosomes by 1 microM Cymelarsan was inhibited by adenosine, adenine and Berenil but not by inosine. Furthermore, in drug-sensitive trypanosomes, adenosine uptake could be reduced in three stages by the sequential addition of inosine, Berenil and adenine, whereas in drug-resistant parasites a reduction in adenosine uptake was caused only by the addition of inosine and adenine. These observations provide evidence that the acquisition of resistance to Cymelarsan is accompanied by the loss of one of three adenosine transporters in T. evansi. In drug-sensitive trypanosomes, this transporter mediates the entry of adenine and also of Cymelarsan and Berenil into the parasites.
The effects of purine starvation on the ability of the trypanosomatid Crithidia luciliae to accumulate purines were determined. Kinetic studies showed that the uptake of the nucleoside adenosine by purine-starved organisms was approximately 7-fold faster than by nutrient-replete cells. Further, these studies demonstrated that purine-starved organisms accumulated the nucleobases hypoxanthine and adenine at a rate > 100-fold faster than organisms cultivated under replete conditions. Activities of several intracellular purine-salvage enzymes were measured in organisms from both culture conditions. Of those measured, the activities of adenine deaminase and hypoxanthine phosphoribosyltransferase were elevated approximately 4-fold and approximately 11-fold, respectively, in purine-starved organisms. Competitive substrate specificity studies suggested that these elevated enzyme activities were not responsible for the increased rates of uptake by purine-starved cells. The results are consistent with the induction of novel surface membrane purine transporters expressed in response to purine starvation. These studies using C. luciliae may provide insights into the mechanisms of trypanosomatid adaptation to altered environments encountered during the course of the life cycle.
Nutritional insufficiency is a common environmental extreme to which parasitic protozoa are routinely exposed. In this study of purine salvage mechanisms we illustrate some successful adaptations of the parasite Crithidia luciliae to its environment, particularly in the case of purine stress. In purine-depleted conditions, the insect trypanosome C. luciliae has the ability to increase the rates of transport of adenosine, guanosine and hypoxanthine and the activity of the exoenzyme 3'nucleotidase (3'NTase) during the growth cycle. The dramatic increase in these activities appears after a 72-h period in culture. The increased activity of the purine transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine or hypoxanthine (100 microM). Under conditions where the concentration of purines in the medium could be closely regulated, C. luciliae grown in purine-replete medium (> or = 75 microM purine) exhibited low rates of purine transport and activity of 3'NTase. In comparison, parasites transferred to medium with a low purine source (< or = 7.5 microM adenosine) had levels of adenosine, guanosine and hypoxanthine transport elevated 25-40-fold. The results link the simultaneous increase in activity of the nucleoside and base transporters, 3'NTase activity and a general increase in the purine salvage of C. luciliae to the concentration of purines available at any time to the parasite.
We have examined cross-resistance between trypanocidal drugs using a well-characterised drug-sensitive line, 247, and its cymelarsan-resistant derivative, 247melCyR. The cymelarsan-resistant line was cross-resistant to trimelarsen and melarsen oxide, and partially cross-resistant to two diamidines, pentamidine and berenil (diminazene aceturate). It was cross-resistant to lipid-soluble melarsoprol in vivo but to only a trivial degree in two in vitro assays. The potential role of adenosine transport in arsenical-induced killing of parasites was investigated. Adenosine, adenine, and the diamidines, but not inosine, were able to inhibit killing of drug-sensitive STIB 247 trypanosomes by cymelarsan and melarsen oxide in a concentration-dependent manner. These results are consistent with the view that these arsenical compounds enter trypanosomes via an adenosine-specific transporter. Melarsoprol-induced killing of trypanosomes was unaffected, however, by either purine and to only a slight degree by the diamidines. These data suggest that melarsoprol can enter trypanosomes by a route other than through an adenosine transporter and that there may be two mechanisms contributing to arsenical resistance in this drug-resistant line of trypanosomes.
The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22 degrees C was rapid and saturable, exhibiting an apparent Km of 9.3 +/- 2.0 microM and a Vmax of 4.5 +/- 0.8 pmol x (10(7) cells)(-1) x s(-1). All the natural purine nucleobases tested (Ki 1.8-7.2 microM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2',7'-bis-(carboxyethyl)-5,6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi), the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH 7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electrogenic. These data show that hypoxanthine uptake in T. b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.
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.
In this paper we describe an approach to selectively deliver compounds to trypanosomes using an adenosine transporter which is unique to the trypanosome. Various polyamine analogues have been attached to known substrates of this adenosine transporter. The compounds prepared interact specifically with the adenosine transporter, some with a similar efficiency to berenil, a known substrate.
Nearly 30 years have passed since purine salvage enzymes were first proposed as targets of drugs in the chemotherapeutic treatment of diseases caused by parasites. The rationale behind a structure-based approach to the design of chemotherapeutic agents involves the use of information about substrate preference and the three-dimensional structure of a target enzyme to design potent selective inhibitors of that enzyme. This approach is outlined here by Syd Craig and Ann Eakin, as it applies to the possible design of inhibitors of a purine salvage enzyme, the hypoxanthine phosphoribosyltransferase.
Parasites of the family Trypanosomatidae have an absolute requirement for purines, yet lack the intracellular machinery to synthesize their own purine ring de novo. As a result, the enzymes devoted to the transport and metabolism of purines are extremely important to the parasite. Here, Claudia Cohn and Michael Gottlieb emphasize the value of understanding purine salvage for the development of trypanocidal drugs, and discuss the putative transporters devoted to purine uptake.
The acquisition of purines by trypanosomatids
- A G Scott
- A Tait
- Cmr Turner
Scott AG, Tait A, Turner CMR. The acquisition of
purines by trypanosomatids. Exp Parasitol 1997;86:181-90.
The acquisition of purines by trypanosomatids
- Scott