Jean-Michel Ubeda

Background: Drug resistance can be complex, and several mutations responsible for it can co-exist 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. Results: Leishmania is a eukaryotic organism with minimal control at the level of transcription initiation and few genes were differentially expressed without concomitant changes in DNA copy number. One exception was found in Leishmania major, where the expression of whole chromosomes was down-regulated. The microarrays highlighted several mechanisms by which the copy number of genes involved in resistance was altered; these include gene deletion, formation of extrachromosomal circular or linear amplicons, and the presence of supernumerary chromosomes. In the case of gene deletion or gene amplification, the rearrangements have occurred at the sites of repeated (direct or inverted) sequences. These repeats appear highly conserved in both species to facilitate the amplification of key genes during environmental changes. When direct or inverted repeats are absent in the vicinity of a gene conferring a selective advantage, Leishmania will resort to supernumerary chromosomes to increase the levels of a gene product. Conclusion: Aneuploidy has been suggested as an important cause of drug resistance in several organisms and additional studies should reveal the potential importance of this phenomenon in drug resistance in Leishmania.

Background: Leishmaniaparasites cause a diverse spectrum of diseases in humans ranging from spontaneously healing skin lesions (e.g.,L. major) to life-threatening visceral diseases (e.g.,L. infantum). The high conservation in gene content and genome organization betweenLeishmania majorandLeishmania infantumcontrasts their distinct pathophysiologies, suggesting that highly regulated hierarchical and temporal changes in gene expression may be involved.Results: We used a multispecies DNA oligonucleotide microarray to compare whole-genome expression patterns of promastigote (sandfly vector) and amastigote (mammalian macrophages) developmental stages betweenL. majorandL. infantum. Seven per cent of the totalL. infantumgenome and 9.3% of theL. majorgenome were differentially expressed at the RNA level throughout development. The main variations were found in genes involved in metabolism, cellular organization and biogenesis, transport and genes encoding unknown function. Remarkably, this comparative global interspecies analysis demonstrated that only 10–12% of the differentially expressed genes were common toL. majorandL. infantum. Differentially expressed genes are randomly distributed across chromosomes further supporting a posttranscriptional control, which is likely to involve a variety of 3'UTR elements.Conclusion: This study highlighted substantial differences in gene expression patterns betweenL. majorandL. infantum. These important species-specific differences in stage-regulated gene expression may contribute to the disease tropism that distinguishesL. majorfromL. infantum.