Project description:The mRNA expression of antimony resistant strains of Leishmania donovani was compared to the expression of the sensitive Leishmania donovani.
Project description:In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression Keywords: stage differentiation; axenic amastigotes
Project description:The mRNA expression of antimony resistant strains of Leishmania donovani was compared to the expression of the sensitive Leishmania donovani. The antimony resistant and sensitive Leishmania donovani were grown in complete M199 medium with 10% FCS and Penicillin streptomycin mixture. At stationary phase (5 day culture) cells were harvested in sterile Phosphate buffered saline and used for RNA isolation.
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. Two condition experiment: NFV-sensitive vs resistant. Biological replicates: Three. One dye swap.
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. Two condition experiment: NFV-sensitive vs resistant. Biological replicates: Three. One dye swap.
Project description:Gene expression analysis was carried out between experimental artesunate resistant Leishmania donovani (adapted to 50 µM artesunate drug pressure) and it's corresponding wild type artesunate sensitive parasite.
Project description:Gene expression analysis was carried out between experimental Paromomycin resistant Leishmania donovani (adapted to 100 µM paromomycin drug pressure) and it's corresponding wild type paromomycin sensitive parasite.