Gene expression modulation is associated with gene amplification, supernumerary chromosomes and chromosome loss in antimony-resistant Leishmania infantum

Abstract

Antimonials remain the first line drug against the protozoan parasite Leishmania but their efficacy is threatened by resistance. We carried out a RNA expression profiling analysis comparing an antimony-sensitive and -resistant (Sb2000.1) strain of Leishmania infantum using whole-genome 70-mer oligonucleotide microarrays. Several genes were differentially expressed between the two strains, several of which were found to be physically linked in the genome. MRPA, an ATP-binding cassette (ABC) gene known to be involved in antimony resistance, was overexpressed in the antimony-resistant mutant along with three other tandemly linked genes on chromosome 23. This four gene locus was flanked by 1.4 kb repeated sequences from which an extrachromosomal circular amplicon was generated in the resistant cells. Interestingly, gene expression modulation of entire chromosomes occurred in the antimony-resistant mutant. Southern blots analyses and comparative genomic hybridizations revealed that this was either due to the presence of supernumerary chromosomes or to the loss of one chromosome. Leishmania parasites with haploid chromosomes were viable. Changes in copy number for some of these chromosomes were confirmed in another antimony-resistant strain. Selection of a partial revertant line correlated antimomy resistance levels and the copy number of aneuploid chromosomes, suggesting a putative link between aneuploidy and drug resistance in Leishmania.

Adaptive mechanisms in pathogens: universal aneuploidy in Leishmania

“…A copy of the targeted gene was always present as a result of chromosome duplications (tri-or tetrasomy), wholegenome duplication, or translocation of the chromosome fragment carrying the target gene. Aneuploidy was also observed in laboratory strains of L. major and L. infantum after induction of drug resistance: transcriptional profiling using microarrays identified altered expression of genes on six different chromosomes in both species [10,11]. Quantitative Southern blot and comparative genomic hybridizations confirmed that the variation in expression corresponded to changes in the copy number of these chromosomes.…”

“…By contrast, chromosome gain or loss (aneuploidy) in multicellular eukaryotes is either lethal or results in severe abnormalities, for example trisomy of chromosome 21 in humans with Down syndrome [6]. Despite the predominantly negative effects of aneuploidy, supernumerary chromosomes have been reported in tumor cells, yeast, and recently the protozoan Leishmania [7][8][9][10][11].…”

“…In C. albicans, aneuploidy can give an adaptive advantage in stressful conditions after only one or two cell divisions, and the original euploid state can be restored in one step when conditions are returned to normal [54]. Reversion to the normal state is also observed in L. infantum, where additional chromosomes disappeared after removal of drug pressure [11]. The generation of aneuploid karyotypes could be achieved by different mechanisms.…”

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

“…3A). Reduced RNA levels in Leishmania are usually due to a decrease in gene copy number (61,62), although other mechanisms may also occur (63). Interestingly, in the yeast S. cerevisiae that also transports AdoMet, resistance to SNF was shown to correlate with loss of function mutations in the AdoMet transporter Sam3 (20).…”

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

“…Although the mode of SbIII's action against Leishmania is poorly understood, mechanisms of resistance have been extensively described and involve the modulation of SbIII detoxifying pathways (36,37), altered uptake (19,38), increased efflux (39), and intracellular sequestration mediated by the ABC-thiol transporter multidrug resistance protein A (MRPA) (37,(40)(41)(42). The Cos-Seq screen for SbIII revealed 11 enriched cosmids ( Fig.…”