Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.

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.

Background: Drug resistance can be complex, and several mutations responsible for it can coexist in a resistant cell. Transcriptional profiling is ideally suited for studying complex resistance genotypes and has the potential to lead to novel discoveries. We generated full genome 70-mer oligonucleotide microarrays for all protein coding genes of the human protozoan parasites Leishmania major and Leishmania infantum. These arrays were used to monitor gene expression in methotrexate resistant parasites.

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.