GENETIC-ANALYSIS OF PURINE METABOLISM IN LEISHMANIA-DONOVANI

To dissect the contributions of hypoxanthine-guanine phosphoribosyltra nsferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenos ine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains la cking either APRT or AK activity, These knockouts were obtained either by double targeted gene replacement or by single gene replacement fol lowed by negative selection for loss-of-heterozygosity. Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorpo rate radiolabeled purine bases, and growth on various purine sources. All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous [H-3]hypoxant hine to the nucleotide level. The surprising ability of mutant L. dono vani lacking HGPRT, APRT, and/or AK to incorporate and grow in hypoxan thine could be attributed to the ability of the parasite xanthine phos phoribosyltransferase enzyme to salvage hypoxanthine. These genetic st udies indicate that HGPRT, APRT, and AK, individually or in any combin ation, are not essential for the survival and growth of the promastigo te stage of L. donovani and intimate an important, if not crucial, rol e for xanthine phosphoribosyltransferase in purine salvage.