We have characterized the genome sequence of five independent in vitro mutants of Leishmania infantum resistant to 5-fluorouracil and pinpointed mutations implicated in resistance.

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines. 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 amplification of DHFR-TS and a deletion of part of chromosome 10 was detected in one mutant. Point mutations in uridine 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 although the identity of the transporter remains elusive. This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.