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ABSTRACT: 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.

PROVIDER: PRJEB1713 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR248703_1.fastq.gz	Fastqsanger.gz
	ERR248703_2.fastq.gz	Fastqsanger.gz
	ERR248704_1.fastq.gz	Fastqsanger.gz
	ERR248704_2.fastq.gz	Fastqsanger.gz
	ERR248705_1.fastq.gz	Fastqsanger.gz

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Comparative genomic hybridization of strains of Leishmania infantum wild-type and five independent 5-fluorouracil resistant mutant.

Project description:The genomic DNAs of strains 263 of L. infantum and five derived independent resistant mutants to 5-fluorouracil were used in comparative genomic hybridizations to reveal the deletion and/or amplification events occured by drug resistance mechanisms. The human protozoan parasites Leishmania are prototrophic for pyrimidines and de novo pyrimidine biosynthesis is necessary for their growth. 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 and deletion events as well as point mutations in metabolic genes involved in either the uridine salvage, folate or dTMP biosynthesis pathways. In particular, a dhfr-ts containing amplicon was observed in two mutants 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 discovered. Transfection experiments confirmed that these molecular alterations were responsible for the 5-FU resistance phenotype. 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 in a cell to lead to resistance. Each independent resistant mutant to 5-fluorouracil was hybridizated with the wild-type L. infantum 263 to 10 microarrays, each with three biological replicates (independent cultures).

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