Point mutations within the LdNT2 nucleoside transporter gene from Leishmania donovani confer drug resistance and transport deficiency.

Leishmania donovani, a protozoan parasite, expresses an unusual inosine/guanosine-specific transporter, LdNT2, the gene for which was cloned by functional rescue of a drug-resistant, LdNT2-deficient (FBD5) strain. In this investigation, we have uncovered and characterized the mutations within the LdNT2 open reading frame that are the basis for the drug-resistance and transport-incompetent phenotype of the FBD5 line. The FBD5 cells were shown to be compound heterozygotes in which both mutant ldnt2 alleles harbor discrete point mutations, each of which impaired transport function and conferred resistance to formycin B, the drug to which the clonal FBD5 line was selected. One of the mutant ldnt2 alleles encoded an S189L alteration in predicted transmembrane domain 5, while the second allele accommodated a null mutation at codon 376, which truncated the transporter just prior to transmembrane domain 8. In addition to the null transport phenotype, very little S189L ldnt2 mutant transporter targeted to the surface of the parasite. The bulk of the truncated ldnt2 appeared to be sequestered internally, possibly within the endoplasmic reticulum, but some of the truncated transporter seemed to be cell surface exposed. The ability to dissect mutations within a viable parasite offers LdNT2 as an attractive model for implementing a thorough forward genetic dissection of transporter function in a eukaryotic cell.

Adenosine kinase from Cryptosporidium parvum.

Analysis of the Cryptosporidium parvum genome demonstrates that the parasite cannot synthesize purines de novo and reveals that the sole route for purine salvage by the parasite is via adenosine kinase (CpAK). In order to initiate a biochemical characterization of CpAK and ultimately validate this apparently essential enzyme as a therapeutic target, the CpAK gene was redesigned for optimum codon usage, overexpressed in Escherichia coli, and the recombinant protein purified to homogeneity and characterized. CpAK appears to be specific for adenosine among the naturally occurring nucleosides but can utilize ATP, GTP, UTP and CTP as the phosphate donor. The enzyme exhibits K(m) values of 1.4microM for adenosine and 41microM for ATP, has a pH optimum approximately 7.0, and is dependent upon the presence of a divalent cation. Structure-activity data intimate that catalysis requires contacts between residues on CpAK with the six-position of the purine ring and the O2' and O3' hydroxyls of the ribose sugar. Additionally, 4-nitro-6-benzylthioinosine, a compound that demonstrates therapeutic promise against the related parasite Toxoplasma gondii, also inhibits adenosine phosphorylation by CpAK. The overproduction and purification of CpAK now enables a thorough evaluation of its potential as a drug target.

Functional characterization of nucleoside transporter gene replacements in Leishmania donovani.

Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine-pyrimidine nucleoside transporter and the LdNT2 inosine-guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity. The mutant alleles were verified by Southern blotting, and the effects of gene replacement on transport phenotype were evaluated by rapid sampling transport measurements and by drug resistance profiles. The Deltaldnt1, Deltaldnt2, and Deltaldnt1/Deltaldnt2 mutants were all capable of proliferation in defined culture medium supplemented with any of a spectrum of purine nucleobases or nucleosides, except that a Deltaldnt2 lesion conferred an inability to efficiently salvage exogenous xanthosine, a newly discovered ligand of LdNT2. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in J774 and bone marrow-derived murine macrophages. These genetic studies demonstrate: (1) that L. donovani promastigotes, axenic amastigotes, and tissue amastigotes are viable in the absence of nucleoside transport; (2) that nucleoside transporters are not essential for sustaining an infection in mammalian host cells; (3) that the phagolysosome of macrophages is likely to contain purines that are not LdNT1 or LdNT2 ligands, i.e., nucleobases. Furthermore, the Deltaldnt1, Deltaldnt2, and Deltaldnt1/Deltaldnt2 knockouts offer a unique genetically defined null background for the biochemical and genetic characterization of nucleoside transporter genes and cDNAs from phylogenetically diverse species and of genetically manipulated LdNT1 and LdNT2 constructs.