Browsing by Author "Marc Ouellette"

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A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death
(2007) Baptiste Vergnes; Benjamin Gourbal; Isabelle Girard; Shyam Sundar; Jolyne Drummelsmith; Marc Ouellette
The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials (Sb(V)), but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory isolates, but our understanding of resistance in field isolates is lagging behind. We describe here a comparative analysis of a genetically related pair of Sb(V)-sensitive and -resistant Leishmania donovani strains isolated from kala-azar patients. The resistant isolate exhibited cross-resistance to other unrelated Leishmania drugs including miltefosine and amphotericin B. A comparative proteomics screen has highlighted a number of proteins differentially expressed suggesting that programmed cell death (PCD) is modified in the resistant parasite. Indeed drug-induced PCD progression was altered in the SbM-resistant strain as determined using early and late markers of apoptosis. Two proteins, the heat shock protein HSP83 and the small kinetoplastid calpain-related protein (SKCRP14.1) were shown to be intimately implicated in the drug-induced PCD phenotype. HSP83 increased drug resistance and reduced drug-mediated PCD activation by interfering with the mitochondrial membrane potential, whereas SKCRP14.1 promoted antimonial-induced PCD but protected against miltefosine-induced PCD. This study highlights the important role of PCD in drug susceptibility/resistance in the protozoan parasite Leishmania. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin
(2004) Benjamin Gourbal; Niluefer Sonuc; Hiranmoy Bhattacharjee; Danielle Legare; Shyam Sundar; Marc Ouellette; Barry P. Rosen; Rita Mukhopadhyay
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.
Intracellular survival of Leishmania species that cause visceral Leishmaniasis is significantly reduced by HIV-1 protease inhibitors
(University of Chicago Press, 2008) Nathalie Trudel; Ravendra Garg; Nadine Messier; Shyam Sundar; Marc Ouellette; Michel J. Tremblay
Visceral leishmaniasis is now recognized as an opportunistic disease in individuals infected with human immunodeficiency virus type 1 (HIV-1). Although the usefulness of HIV-1 protease inhibitors (PIs) in antiretroviral regimens is well documented, little is known about their potential impact in the setting of Leishmania/HIV-1 coinfections. We now report that, although selected PIs do not inhibit the growth of Leishmania infantum promastigotes alone in culture, these drugs significantly inhibit the intracellular survival of parasites in phorbol myristate acetate-differentiated THP-1 macrophages and human primary monocyte-derived macrophages (MDMs). Furthermore, a field isolate of Leishmania donovani resistant to sodium stibogluconate (SbV), one of the drugs most commonly used to treat leishmaniasis, is equally susceptible to the tested PIs compared with a sensitive strain, thus suggesting that resistance to SbV does not result in cross-resistance to PIs. Importantly, the efficacy of PIs to reduce the intracellular growth of Leishmania parasites is also observed in MDMs coinfected with HIV-1. © 2008 by the Infectious Diseases Society of America. All rights reserved.
Mutations in an Aquaglyceroporin as a Proven Marker of Antimony Clinical Resistance in the Parasite Leishmania donovani
(Oxford University Press, 2021) Jade-Eva Potvin; Philippe Leprohon; Marine Queffeulou; Shyam Sundar; Marc Ouellette
Background: Antimonial drugs have long been the mainstay to treat visceral leishmaniasis. Their use has been discontinued in the Indian subcontinent because of drug resistance, but they are still clinically useful elsewhere. The goal of this study was to find markers of antimony resistance in Leishmania donovani clinical isolates and validate experimentally their role in resistance. Methods: The genomes of sensitive and antimony-resistant clinical isolates were sequenced. The role of a specific gene in contributing to resistance was studied by CRISPR-Cas9-mediated gene editing and intracellular drug sensitivity assays. Results: Both gene copy number variations and single nucleotide variants were associated with antimony resistance. A homozygous insertion of 2 nucleotides was found in the gene coding for the aquaglyceroporin AQP1 in both resistant isolates. Restoring the wild-type AQP1 open reading frame re-sensitized the 2 independent resistant isolates to antimonials. Alternatively, editing the genome of a sensitive isolate by incorporating the 2-nucleotide insertion in its AQP1 gene led to antimony-resistant parasites. Conclusions: Through genomic analysis and CRISPR-Cas9-mediated genome editing we have proven the role of the AQP1 mutations in antimony clinical resistance in L. donovani. © 2020