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.
Project description:In this study we report the complete repertoire of 2'-O-methylation sites present in the rRNA of Leishmania amazonensis rRNAs and a subset of small RNAs using RibOxi-seq.
Project description:To determine the modulation of gene expression of C57BL/6 and DBA/2 BMDLs in the presence of living intracellular Leishmania amazonensis amastigotes
Project description:To determine the modulation of gene expression of C57BL/6 and DBA/2 BMDLs in the presence of living intracellular Leishmania amazonensis amastigotes A genome-wide transcriptional analysis was performed by comparing the gene expression profiles of control DLs and live amastigote-hosting DLs from both mouse strains. Dendritic Leucocytes were generated in vitro from bone marrow progenitors (C57Bl/6 and DBA/2 mice). Leishmania amazonensis amastigotes were purified from mouse cutaneous lesions and were added to DL cultures. After 24h, and following a sorting procedure, only BMDls housing living amastigotes were selected for total RNA extraction. Three Biological replicates per condition were run.
Project description:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes
Project description:Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. (L.) infantum and L. (L.) amazonensis are causative agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts. However, this approach tends to underrepresent membrane-associated proteins because of their high hydrophobicity and low solubility. Due to the great importance of membrane-associated proteins in biological processes, including host–parasite interactions, virulence and invasiveness, this study applied label-free shotgun proteomics to characterize and evaluate abundance levels of plasma membrane sub-proteome of promastigotes life-stage. The total number of proteins identified in L. (L.) infantum and L. (L.) amazonensis was 2033 and 2243, respectively. Both species shared 1908 of these quantified proteins. After cell localization prediction of all identified proteins, 394 proteins were described as plasma membrane-associated proteins and their majority (320 proteins) was presented in both species. Considering only exclusive proteins, 18 proteins were detected only in L. (L.) infantum and 56 proteins in L. (L.) amazonensis. We used two criteria to define “regulated” proteins; i) proteins with p-value < 0.05 after One-Way ANOVA analysis (quantitative analysis) and proteins detected only in L. (L.) infantum or L. (L.) amazonensis (qualitative analysis).
Project description:Skin from Balb/c mice after Leishmania amazonensis infection. Mice were infected through their footpads with the promastigote form of the protozoon
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:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes Dendritic cells were generated in vitro from bone marrow progenitors. Leishmania amazonensis amastigotes were purified from mouse cutaneous lesions and were added to DC cultures. After 24h, and following a sorting procedure, only BMDCs housing living amastigotes were selected for total RNA extraction.