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.
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).
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.
Project description:Comparative genomic hybridization of strains of Leishmania infantum wild-type and five independent 5-fluorouracil resistant mutant.
Project description:To investigate dendritic cells-Leishmania interaction, the transcriptional profile of bone marrow-derived dendritic cells (BMDCs) infected with Leishmania infantum or of cells exposed to chemically inactivated parasites was assessed
Project description:Transcriptional analyses of L. infantum promastigote compared to L. infantum intracellular amastigote, and L. major promastigote compared to L. major intracellular amastigote The full-genome DNA microarray includes one 70mer-oligonucleotide probe for each gene of L. infantum and for each gene of L.major LV39 Keywords: stage-specific comparison Leishmania infantum: Two-condition experiment, promastigote stage vs amastigote stage. Six biological replicates for each stage, independently grown and harvested. One replicate per array Leishmania major: Two-condition experiment, promastigote stage vs amastigote stage. Four biological replicates for each stage, independently grown and harvested. One replicate per array
Project description:The uploaded model is linked to the PLoS ONE article: Subramanian A, Jhawar J, Sarkar RR (2015) Dissecting Leishmania infantum Energy Metabolism - A Systems Perspective. PLoS ONE 10(9): e0137976. https://doi.org/10.1371/journal.pone.0137976. The reconstructed network consists of 142 genes encoding for enzymes performing 237 reactions distributed across five distinct model compartments. We annotated the subcellular locations of different enzymes and their reactions on the basis of strong literature evidence and sequence-based detection of cellular localization signal within a protein sequence. We also predicted an extensive set of lethal reaction knockouts; some of which were validated using published data on Leishmania species. Performing a robustness analysis, the model was rigorously validated and tested for the secretion of overflow metabolites specific to Leishmania under varying extracellular oxygen uptake rate. Stage-specific scenarios of L. infantum energy metabolism were incorporated in the model and key metabolic differences were outlined. Analysis of the model revealed the essentiality of glucose uptake, succinate fermentation, glutamate biosynthesis and an active TCA cycle as driving forces for parasite energy metabolism and its optimal growth. Finally, through our in silico knockout analysis, we could identify possible therapeutic targets that provide experimentally testable hypotheses.
Project description:This SuperSeries is composed of the following subset Series: GSE9947: Transcriptional analysis of Leishmania infantum methotrexate resistant strains using full-genome DNA microarrays GSE9948: Transcriptional analysis of Leishmania major methotrexate resistant strains using full-genome DNA microarrays Keywords: SuperSeries Refer to individual Series
Project description:Gene expression profiling to address the effects of infection with Leishmania infantum during distinct clinical outcomes as active visceral leishmaniasis (VL), remission of disease and asymptomatic infection.
2016-10-25 | GSE77528 | GEO
Project description:MRPA-independent mechanisms of antimony resistance in Leishmania infantum