Nicola S. Carter

Nucleoside and Nucleobase Transporters in Parasitic Protozoa

One distinctive feature of the biochemistry of parasitic protozoa is their absolute reliance upon the salvage of preformed purines from their vertebrate and invertebrate hosts. While many mammalian cells possess the innate ability to synthesize purines de novo, all protozoa so far examined that

Characterisation of melarsen-resistant Trypanosoma brucei brucei with respect to cross-resistance to other drugs and trypanothione metabolism

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of Δodccells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The Δodc L. donovaniexhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the Δodc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.

Cloning of a novel inosine-guanosine transporter gene from Leishmania donovani by functional rescue of a transport-deficient mutant.

Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovanideficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2re-established their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K m = 0.3 μm) and guanosine (K m = 1.7 μm) and does not recognize other naturally occurring nucleosides. Expression ofLdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.

Genetic analysis of spermidine synthase from Leishmania donovani.

The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Deltaspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Deltaspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.

S-adenosylmethionine decarboxylase from Leishmania donovani: Molecular, genetic, and biochemical characterization of null mutants and overproducers

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced fromLeishmania donovani. The Δadometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Δadometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Δadometdcparasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomalADOMETDC construct were created in both wild type and Δadometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Δadometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5′-(((Z)-4-amino-2-butenyl)methylamino)-5′-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.

Purine and Pyrimidine Metabolism in Leishmania

Purines and pyrimidines are indispensable to all life, performing many vital functions for cells: ATP serves as the universal currency of cellular energy, cAMP and cGMP are key second messenger molecules, purine and pyrimidine nucleotides are precursors for activated forms of both carbohydrates and lipids, nucleotide derivatives of vitamins are essential cofactors in metabolic processes, and nucleoside triphosphates are the immediate precursors for DNA and RNA synthesis. Unlike their mammalian and insect hosts, Leishmania lack the metabolic machinery to make purine nucleotides de novo and must rely on their host for preformed purines. The obligatory nature of purine salvage offers, therefore, a plethora of potential targets for drug targeting, and the pathway has consequently been the focus of considerable scientific investigation. In contrast, Leishmania are prototrophic for pyrimidines and also express a small complement of pyrimidine salvage enzymes. Because the pyrimidine nucleotide biosynthetic pathways of Leishmania and humans are similar, pyrimidine metabolism in Leishmania has generally been considered less amenable to therapeutic manipulation than the purine salvage pathway. However, evidence garnered from a variety of parasitic protozoa suggests that the selective inhibition of pyrimidine biosynthetic enzymes offers a rational therapeutic paradigm. In this chapter, we present an overview of the purine and pyrimidine pathways in Leishmania, make comparisons to the equivalent pathways in their mammalian host, and explore how these pathways might be amenable to selective therapeutic targeting.

Characterisation of pentamidine-resistant Trypanosoma brucei brucei.

Following selection in vitro by exposure to increasing concentrations of the aromatic diamidine pentamidine, a Trypanosoma brucei brucei clone has been characterised in vivo and in vitro. The resistant clone, designated T.b. brucei S427/118/PR32.6, was found to be less virulent than the parental clone T.b. bruci S427/118, with an intraperitoneal injection of 2.5 x 10(6) resistant organisms required to produce a course of disease equivalent to 1 x 10(4) sensitive trypanosomes. This lowered virulence is not associated with an increased susceptibility to the hosts immune system, and is not due to the in vitro culturing process. The pentamidine-resistant clone was found to be 26- and 4.5-fold resistant to pentamidine in vitro and in vivo, respectively. Although not cross-resistant in vivo to any other aromatic diamidines (stilbamidine, berenil and propamidine), a 2.4-fold increase in resistance to the melaminophenylarsine melarsoprol was observed. While pentamidine completely inhibited uptake of 1 microM [3H]adenosine in the presence of 1 mM inosine, suggesting that pentamidine is transported by the inosine-insensitive P2 transporter, the pentamidine-resistant clone appeared to have a fully functional P2-adenosine transport system. Both resistant and parental cloned lines accumulated approx. 6 nmol pentamidine (10(8) cells)-1 over the course of 3 h, representing an internal concentration of 0.7-1.0 mM. Thus, unlike previously characterised drug-resistant trypanosomes, T.b. brucei PR32.6 is not deficient in drug accumulation, suggesting that other resistance mechanisms are likely to be involved.

Nucleoside transporters of parasitic protozoa.

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.

Cloning and Functional Expression of a Gene Encoding a P1 Type Nucleoside Transporter from Trypanosoma brucei

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purinesde novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of theTbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites.TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.