title: Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes.
abstract: Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations 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. Microarray analyses of Nelfinavir-resistant and -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. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles.
authors.affiliation: Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec - CHUL, Québec, Canada, G1V 4G2.
authors.forename: Pranav
authors.initials: P
authors.lastname: Kumar
authors.forename: Robert
authors.initials: R
authors.lastname: Lodge
authors.forename: Frédéric
authors.initials: F
authors.lastname: Raymond
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Ritt
authors.forename: Pascal
authors.initials: P
authors.lastname: Jalaguier
authors.forename: Jacques
authors.initials: J
authors.lastname: Corbeil
authors.forename: Marc
authors.initials: M
authors.lastname: Ouellette
authors.forename: Michel J
authors.initials: MJ
authors.lastname: Tremblay
mesh_terms: D000782:Aneuploidy; D002478:Cells, Cultured; D055028:Comparative Genomic Hybridization; D056915:DNA Copy Number Variations; D004351:Drug Resistance; D020869:Gene Expression Profiling; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects / Q000235:genetics*; D008264:Macrophages / Q000469:parasitology; D019888:Nelfinavir / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D016053:RNA, Protozoan / Q000235:genetics; D015854:Up-Regulation
chemical_list: D016053:RNA, Protozoan; D019888:Nelfinavir