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Adenine phosphoribosyltransferase-deficient Leishmania donovani
Abstract:
Mutant promastigotes of Leishmania donovani deficient in adenine phosphoribosyltransferase (APRTase) have been isolated in medium containing 4-aminopyrazolopyrimidine. The generation of APRTase-deficient mutants occurred in two discrete steps. In the first step, clones were isolated with 50% of wildtype levels of APRTase activity. These cells were reselected and colonies totally deficient in APRTase were isolated. Partially and totally APRTase-deficient cells exhibited intermediate and complete resistance to cytotoxic adenine analogs, respectively. Nevertheless, wildtype and mutant cells could salvage adenine and utilize adenine as a purine source equally efficiently, suggesting that the adenine deaminase-HGPRTase pathway plays an important role in promastigote adenine metabolism. Kinetic and thermal inactivation studies of purified APRTase and isoelectric focusing of crude extracts from wildtype and partially APRTase-deficient cells suggested that the latter cells possessed wildtype APRTase activity at half the amount found in wildtype parental cells. These data suggest that Leishmania donovani possess two copies of the APRTase structural gene and that these organisms might be diploid for the APRTase locus.
Insights
Leishmania donovani mutants deficient in adenine phosphoribosyltransferase (APRTase) were generated in two steps. These mutants showed resistance to cytotoxic adenine analogs, suggesting a diploid genome for the APRTase locus.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Leishmania donovani is a parasite responsible for visceral leishmaniasis.
- Adenine phosphoribosyltransferase (APRTase) is crucial for purine salvage pathways in many organisms.
- Understanding purine metabolism in Leishmania is vital for developing novel therapeutic strategies.
Purpose of the Study:
- To generate and characterize adenine phosphoribosyltransferase (APRTase)-deficient mutants of Leishmania donovani.
- To investigate the role of the adenine deaminase-HGPRTase pathway in adenine metabolism.
- To explore the gene copy number of APRTase in Leishmania donovani.
Main Methods:
- Selection of APRTase-deficient mutants using 4-aminopyrazolopyrimidine.
- Assessing resistance to cytotoxic adenine analogs.
- Enzyme kinetics and thermal inactivation studies of APRTase.
- Isoelectric focusing of crude extracts.
Main Results:
- Two-step generation of partially and totally APRTase-deficient mutants.
- Mutants exhibited dose-dependent resistance to adenine analogs.
- The adenine deaminase-HGPRTase pathway plays a significant role in adenine salvage.
- Evidence suggests Leishmania donovani may be diploid for the APRTase gene locus.
Conclusions:
- APRTase deficiency confers resistance to cytotoxic adenine analogs in Leishmania donovani.
- The findings support the hypothesis of diploidy for the APRTase locus in these parasites.
- This study provides insights into purine metabolism and potential drug targets in Leishmania.