Project description:We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. This experiment appears as Fig. 1 of the associated publication. Keywords: RNA expression profiling
Project description:We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance. Keywords: RNA expression profiling
Project description:Gene expression profiling to address the effects of infection with Leishmania infantum during distinct clinical outcomes as active visceral leishmaniasis (VL), remission of disease and asymptomatic infection.
Project description:Drug resistance is a major public health challenge in Leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. Recently, we have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to investigate the underlying molecular mechanism involved in Nelfinavir resistance, in vitro Nelfinavir resistant amastigotes were developed by direct drug pressure in culture. RNA expression profiling analyses of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance, and several genes were found to be differentially expressed. Comparative gene hybridization (CGH) analyses of Nelfinavir-resistant and -sensitive Leishmania using whole-genome 60-mer oligonucleotide microarrays were also carried out. RNA expression profiles and the CGH of Nelfinavir resistant vs sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters, which are involved in vesicular trafficking. Transporter assays using radiolabeled Nelfinavir suggest that the drug accumulates in greater amounts in the resistant parasites and in a time dependent manner. Furthermore, high-resolution electron microscopy showed an increased number of vacuoles in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in these intracellular vesicles.
Project description:Drug resistance is a major public health challenge in Leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. Recently, we have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to investigate the underlying molecular mechanism involved in Nelfinavir resistance, in vitro Nelfinavir resistant amastigotes were developed by direct drug pressure in culture. RNA expression profiling analyses of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance, and several genes were found to be differentially expressed. Comparative gene hybridization (CGH) analyses of Nelfinavir-resistant and -sensitive Leishmania using whole-genome 60-mer oligonucleotide microarrays were also carried out. RNA expression profiles and the CGH of Nelfinavir resistant vs sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters, which are involved in vesicular trafficking. Transporter assays using radiolabeled Nelfinavir suggest that the drug accumulates in greater amounts in the resistant parasites and in a time dependent manner. Furthermore, high-resolution electron microscopy showed an increased number of vacuoles in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in these intracellular vesicles.
Project description:The aim of this study was to identify differences in the NK-cell response towards Leishmania mexicana lipophosphoglycan (LPG) between patients with localized (LCL) and diffuse (DCL) cutaneous leishmaniasis through gene expression profiling, in an attempt to pinpoint alterations in the signaling pathways responsible for the NK-cell dysfunction in patients with DCL. To determine the gene expression profiling in non stimulated and LPG-stimulated NK cells we include samples of controls, LCL and DCL patients. We performed microarrays (Human Gene 1.0 ST, Affymetrix) to identify differentially expressed transcripts between non stimulated and LPG-stimulated NK cells between controls, LCL and DCL samples.
Project description:Murine bone marrow derived macrophages were infected with Leishmania major or Leishmania donovania promastigotes, allowed to phagocytose latex beads or not treated. Gene expression profiles were compared to identify i) the effect of Leishmania infection; ii) the differences in effects between L. major and L. donovani; and iii) the effect of pahgocytosis of latex beads.