Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum |
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| Institution: | 1. IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal;2. Laboratory of Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, CP 11400 Montevideo, Uruguay;3. Departamento de Bioquímica, Universidad de la República, CP 11800 Montevideo, Uruguay, and Department of Molecular Medicine, Università degli Studi di Padova, 35131 Padova, Italy;4. German Center for Infection Research, 38124 Braunschweig, Germany;5. Schirm GmbH, D-39218 Schönebeck, Germany;6. ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4099-003 Porto, Portugal;1. Department of Medicine, Weill Cornell Medical College, New York, NY, USA;2. Departments of Medicine, Molecular Genetics and Microbiology and Immunology, Division of Geriatrics and Center for the Study of Aging and Human Development, Duke University Medical Center, Durham, NC, USA;3. Geriatric Research, Education and Clinical Center, VA Medical Center, Durham, NC, USA;4. Shanghai Jiaotong University, School of Life Science and Biotechnology, Shanghai, China;5. Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY, USA;1. Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA;1. Instituto de Parasitología y Biomedicina ‘López-Neyra’, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento, s/n 18016-Armilla (Granada), Spain;2. Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium;1. Aix Marseille Univ, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin, CS30064, 13385, Marseille Cedex 05, France;2. Université de Limoges, UMR Inserm 1094, Neuroépidémiologie Tropicale, Faculté de Pharmacie, 2 Rue Du Dr Marcland, 87025, Limoges, France;3. Université de Limoges, BISCEm, US 042 INSERM – UMS 2015 CNRS, Mass Spectrometry Platform, CBRS, 2 Rue Du Pr. Descottes, F-87025, Limoges, France;4. Aix Marseille Univ, IHU Méditerranée Infection, UMR VITROME - Tropical Eukaryotic Pathogens, 19-21 Boulevard Jean Moulin, 13005, Marseille, France;5. LCC-CNRS Université de Toulouse, CNRS, UPS, Toulouse, France;6. UMR 152 PHARMA-DEV, Université de Toulouse, IRD, UPS, Toulouse, France;7. Department of Pharmacology and Toxicology, Faculty of Pharmacy and Nutrition, University of Navarra, C/ Irunlarrea 1, CP 31008, Pamplona, Navarra, Spain;8. Department of Pharmacology, Toxicology and Pharmacovigilance, CHU Limoges, Limoges, France;9. INSERM, UMR 1248, University of Limoges, France;10. University of Dundee, School of Life Sciences, Division of Biological Chemistry and Drug Discovery, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom;11. Normandie Univ, UNICAEN, CERMN, 14000, Caen, France;12. Institut Méditerranéen de Biodiversité et D’Ecologie Marine et Continentale (IMBE), Aix-Marseille Université, UMR CNRS IRD Avignon Université, Campus Timone – Faculté de Pharmacie, 27 Boulevard Jean-Moulin, F13385, Marseille Cedex 05, France;1. Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, CP 11400, Montevideo, Uruguay;2. Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, CP 11400, Montevideo, Uruguay;3. BioMolecular Simulation Group, Institut Pasteur de Montevideo, Mataojo 2020, CP 11400, Montevideo, Uruguay |
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| Abstract: | Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps?/? knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys?/?/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage. |
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| Keywords: | Trypanothione Glutathionylspermidine Target validation Free radicals |
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