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: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:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards. Six strains of three Leishmania species were hybridizated to 12 microarrays, each with four biological replicates (independent cultures). Supplementary file: Represents final results obtained after statistical analysis of all replicates.
Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards.
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:Whole genome high density tiliing array of eight timepoints from the promastitgote to amastigote differentiation process. RNA from two independent biological replicates from eight points in the promastigote to amastigote differentiation process were hybridized to Nimblegen arrays (Madison,WI USA) that contained 10 probes per open reading frame and 3 probes per structural RNA spotted three times per array. The microarray probes were designed to Leishmania infantum strain JCPM5 and the sample RNA was obtained from Leishmania donovani strain S1. The two species are thought to be nearly identical at the nucleotide level, however whole-genome sequence was not available from L. donovani.
Project description:To investigate the effect of infection of osteoclast by Leishmania infantum, we infected diffentiating bone marrow derived osteoclast with the parasite at day 2 of differentiation We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cultures of cells infected for 24 hours compared to 4 control cultures.
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.
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