Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

BACKGROUND: The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines. METHODOLOGY/PRINCIPAL FINDINGS: Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import. CONCLUSION/SIGNIFICANCE: This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.