Antimony resistance in Leishmania guyanensis
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REPOSITORIES: ENA
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Project description:Background: Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis. Methods/Principal Findings: We selected populations of L. amazonensis for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated. Conclusions/Significance: Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.
2011-06-11 | GSE26159 | GEO
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.
2023-04-04 | GSE173848 | GEO
Project description:The mRNA expression of antimony resistant strains of Leishmania donovani was compared to the expression of the sensitive Leishmania donovani. The antimony resistant and sensitive Leishmania donovani were grown in complete M199 medium with 10% FCS and Penicillin streptomycin mixture. At stationary phase (5 day culture) cells were harvested in sterile Phosphate buffered saline and used for RNA isolation.
2016-07-03 | E-GEOD-62468 | biostudies-arrayexpress
Project description:The mRNA expression of antimony resistant strains of Leishmania donovani was compared to the expression of the sensitive Leishmania donovani.
2016-01-01 | GSE62468 | GEO
Project description:The aim of the experiment is to detect any gene expression modulation in an antimony resistant L.infantum Sb2000.1 strain compared to an antimony sensitive L.infantum WT strain. We want to identify the modulated genes associated with an antimony resistant phenotype. The Sb2000.1 resistant strain used is about 1000 times more resistant to SbIII than the WT sensitive strain. The full-genome DNA microarrays includes one 70-oligonucleotide probe for each gene of L. infantum. Keywords: drug resistance
2009-08-15 | GSE16965 | GEO
Project description:The aim of the experiment is to detect any gene expression modulation in an antimony resistant L.infantum Sb2000.1 strain compared to an antimony sensitive L.infantum WT strain. We want to identify the modulated genes associated with an antimony resistant phenotype. The Sb2000.1 resistant strain used is about 1000 times more resistant to SbIII than the WT sensitive strain. The full-genome DNA microarrays includes one 70-oligonucleotide probe for each gene of L. infantum. Keywords: drug resistance Two-condition experiment, resistant strain Sb2000.1 vs wild-type. Five biological replicates for each strain, independently grown and harvested. One replicate per array
2009-08-15 | E-GEOD-16965 | biostudies-arrayexpress
Project description:Infection with antimony resistant (SbR) but not with sentitive (SbS) Leishmania donovani (LD) gives rise to aggressive pathology and higher organ parasite load in infected patients, the cause of which is far from clear. The pentose phosphate pathway (PPP) is a key route of glucose metabolism in most organisms, providing NADPH for use as a cellular reductant and various carbohydrate intermediates used in cellular metabolism. Alteration in PPP is primary reason behind aggressive pathology and multi drug resistance in several types of carcinoma and other infectious disease model. A time kinetic study with equal inoculum of SbRLD and SbSLD promastigotes and host cells infected with equal number of SbR and SbSLD, resulted in remarkable higher growth rate in SbRLD which is also reflected in terms of their DNA content. Our result showed an activated PPP pathway in response to oxidative stress in SbRLD. Our result suggest that SbRLD have adapted to a higher nucleotide metaboism that provide them a selective survival advantage in presence of drug mediated oxidative stress and explains, in part, the cause of higher organ parasite load in SbRLD infected patients.
2020-02-04 | GSE144659 | GEO
Project description:Leishmania guyanensis CL085 genome assembled from short read data provided by the Sanger Institute
| PRJEB20208 | ENA
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
2013-05-01 | GSE41750 | GEO