Translational Reprogramming as a Driver of Antimony-drug Resistance in Leishmania

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. Overall design: Three experimental conditions were considered to estimate changes at the translational level: WT strain growing without drug challenge (WT w/o SbIII), HR strain growing without drug challenge (HR w/o SbIII), and HR strain growing under drug challenge (HR + SbIII). Four types of samples were evaluated: total mRNA used as input for polysome profiling. After polysome profiling and automatic fractionation the mRNAs under translation were grouped into three additional categories representing increasing levels of translational efficiency: monosomes (MS), light polysomes (LP), and heavy polysomes (HP). Each experimental condition was evaluated in three biologically independent experiments. As a result, 36 samples (3 conditions X 4 types of samples X 3 biological replicates) were submitted to sequencing in Illumina Hiseq 2000 platform, with read length of 150 nt, and an estimated of 40M PE reads per sample (20M in each direction). After sequencing, three different comparisons were evaluated by differential gene expression analysis. Comparison 1: changes at the translational level in drug-resistant strain occurring in the absence of the drug (general basal changes. HR Vs. WT). Comparison 2: Changes occurring when the resistant parasites are growing under SbIII exposure (changes to combat the drug, HR+SbIII Vs. HR). Comparison 3: Changes occurring at the transcriptional or at the translational level (HP Vs. total mRNA).