Project description:SbIII clonal mutants and an isogenic WT clonal line. Genomic DNA from clonal WT or mutants were digested and hybridized to whole genome DNA microarrays. Antimonials are still the mainstay of treatment against Leishmaniasis but in the past decade resistance has been a severe threat. We carried out short read next generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony resistant mutants. Copy number variations were consistently detected in both NGS and CGH where several chromosomal aneuploidies were correlated to antimony resistance. A major attribute of antimony resistance was a novel terminal deletion of variable length (67kb-204kb) of the polyploid chromosome 31 in the three mutants and was experimentally validated. Terminal deletion in two mutants occurred at the level of inverted repeated sequences in chromosome 31. AQP1 (LmjF.31.0020), a gene encoding for an aquaglyceroporin, which facilitates uptake of trivalent metalloids, was a part of the deleted region. Transfection of AQP1 into resistant mutants rendered them hypersensitive to SbIII. CGH, NGS and Southern blot analysis also highlighted a novel stable, intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded redox enzymes like ascorbate dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH) and overexpression of the genes coding for these enzymes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS) accumulation. Generation of G6PDH null mutant in one revertant exhibited SbIII sensitivity and protection from ROS which were rescued in the add back. Our genomic analyses and parallel functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania. This submission represents the microarray component of the study 3 biological replicates of each sample

Project description:SbIII clonal mutants and an isogenic WT clonal line. Genomic DNA from clonal WT or mutants were digested and hybridized to whole genome DNA microarrays. Antimonials are still the mainstay of treatment against Leishmaniasis but in the past decade resistance has been a severe threat. We carried out short read next generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony resistant mutants. Copy number variations were consistently detected in both NGS and CGH where several chromosomal aneuploidies were correlated to antimony resistance. A major attribute of antimony resistance was a novel terminal deletion of variable length (67kb-204kb) of the polyploid chromosome 31 in the three mutants and was experimentally validated. Terminal deletion in two mutants occurred at the level of inverted repeated sequences in chromosome 31. AQP1 (LmjF.31.0020), a gene encoding for an aquaglyceroporin, which facilitates uptake of trivalent metalloids, was a part of the deleted region. Transfection of AQP1 into resistant mutants rendered them hypersensitive to SbIII. CGH, NGS and Southern blot analysis also highlighted a novel stable, intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded redox enzymes like ascorbate dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH) and overexpression of the genes coding for these enzymes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS) accumulation. Generation of G6PDH null mutant in one revertant exhibited SbIII sensitivity and protection from ROS which were rescued in the add back. Our genomic analyses and parallel functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania. This submission represents the microarray component of the study

Project description:Purpose: In this study, we have used a translatomic approach by coupling polysome profiling and deep RNA-sequencing to estimate changes in the translatome of antimony-resistant Leishmania parasites Methods: Leishmania tropica promastigotes were stepwise selected for resistance to trivalent antimony. Two different strains were studied, the L. tropica SbIII-sensitive or wildtype strain (WT) and the derived highly resistant strain (HR). After polysome profiling four types of samples were evaluated by deep RNAseq: total mRNA used as input, monosomes (MS), light polysomes (LP), and heavy polysomes (HP). The DESeq2 algorithm was used for differential expression analysis to identify translational changes at the basal level (HR Vs. WT), translational changes to combat the drug (HR+SbIII Vs. HR), and to compare translatomic Vs. transcriptomic changes (HP Vs. Total input) [see overall design section below]. Results: Differential translational analysis (cutoff of fold change ≥ 1.5 and p-value corrected by Benjamini-Hochberg FDR ≤ 0.05) showed that transcripts composition per polysome fraction was different in the resistant strain. It included several upregulated (Up) and downregulated (Down) transcripts. At the basal level, 2431 different transcripts were differentially translated: monosome (Down: 4, Up: 0), light polysomes (Down: 906, Up: 951), and heavy polysomes (Down: 1096, Up: 1064). Under the antimony challenge, 189 different transcripts were differentially translated: monosome (Down: 0, Up: 2), light polysomes (Down: 9, Up: 57), and heavy polysomes (Down: 30, Up: 134). Overall, most of the changes were identified in polysome fraction when compared with monosomes or total transcriptome. Conclusions: Our study shows evidence that translational control has a main role in coordinating the resistance to antimony in Leishmania parasites. We propose a novel model that establishes translational control as a major driver of antimony-resistant phenotypes in Leishmania parasites.

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.

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