Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs

Abstract

Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (SbIII, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.

Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

“…Similar results were observed following the overexpression of BCAT, although there was no additive effect when BCAT and D-LDH were coexpressed in the same line. The authors proposed that the enzymatic modification of amino or hydroxyl groups on the antibiotics could contribute to the resistance mechanisms of BCAT and D-LDH (15). In the present study, the levels of 2-keto-3-methylvaleric acid (KMVA), the 2-keto acid derivative of isoleucine, indicated increased BCAT activity in the Sb i PMM r strain compared to that in the corresponding WT.…”

“…Leishmania species do not encode LDH, but genes for malate dehydrogenases (MDH) and a putative D-lactate dehydrogenase (D-LDH) are present. Recently, the upregulation of D-LDH and BCAT mRNA was linked to PMM r in L. donovani by transcriptome sequencing (RNA-seq) analysis of WT and PMM r strains (15). Overexpression of the putative D-LDH in L. donovani, L. major, and L. infantum also increased PMM resistance, and there was cross-resistance with other aminoglycosidic antibiotics.…”

“…However, the product of these gene sequences was unknown and Cos-Seq could not identify loss-of-function targets (14). Previous work showed that the molecular mechanisms of drug resistance may vary with the genetic background of the parasite (15). In the present study, we thus selected for PMM r in three L. donovani clinical strains from Nepal with different susceptibilities to Sb drugs and different genetic backgrounds.…”

Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance

“…A proteomic approach was elected to address these issues in the present study. Although RNA sequencing and transcriptomic analysis have been extensively used to explore molecular alterations that might be associated with drug resistance in Leishmania species [29], such methodology was not employed in this work. This is because post-transcriptional regulations (via microRNAs and other non-coding small RNAs) appear to substantially change the genetic information processing from messenger RNA to protein [45,46].…”

Frequent Recombination Events in Leishmania donovani: Mining Population Data

“…In the current study we focused on a large-scale analysis of putative gene conversion events using publicly available datasets. Previous population study of L. donovani suggested that population variability of L. donovani is fairly low, however the authors used masking procedures and strict read selection criteria [14,32]. We decided to reanalyze DNA-seq data without masking sequences, because we were interested in the most dynamic fraction of the genome.…”