title: Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant.
abstract: Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.
authors.affiliation: Unité de Parasitologie Moléculaire et Signalisation, Institut Pasteur and Institut National de la Santé et de la Recherche Médicale INSERM U1201, 25 rue du Dr Roux, F-75015, Paris, France.|Clos Group (Leishmanasis), Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Street 74, D-20359, Hamburg, Germany.
authors.forename: Wai-Lok
authors.initials: WL
authors.lastname: Yau
authors.affiliation: Division of Infectious Diseases, Jack Bell Research Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia, V6H 3Z6, Canada.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Lambertz
authors.affiliation: Division of Infectious Diseases, Jack Bell Research Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia, V6H 3Z6, Canada.
authors.forename: Lucie
authors.initials: L
authors.lastname: Colineau
authors.affiliation: Unité de Parasitologie Moléculaire et Signalisation, Institut Pasteur and Institut National de la Santé et de la Recherche Médicale INSERM U1201, 25 rue du Dr Roux, F-75015, Paris, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Pescher
authors.affiliation: Clos Group (Leishmanasis), Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Street 74, D-20359, Hamburg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: MacDonald
authors.affiliation: Clos Group (Leishmanasis), Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Street 74, D-20359, Hamburg, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Zander
authors.affiliation: Electron Microscopy Service, Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Street 74, D-20359, Hamburg, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Retzlaff
authors.affiliation: Clos Group (Leishmanasis), Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Street 74, D-20359, Hamburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Eick
authors.affiliation: Division of Infectious Diseases, Jack Bell Research Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia, V6H 3Z6, Canada.
authors.forename: Neil E
authors.initials: NE
authors.lastname: Reiner
authors.affiliation: Clos Group (Leishmanasis), Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Street 74, D-20359, Hamburg, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Clos
authors.affiliation: Unité de Parasitologie Moléculaire et Signalisation, Institut Pasteur and Institut National de la Santé et de la Recherche Médicale INSERM U1201, 25 rue du Dr Roux, F-75015, Paris, France. gerald.spaeth@pasteur.fr.
authors.forename: Gerald F
authors.initials: GF
authors.lastname: Späth
mesh_terms: D000818:Animals; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000172:deficiency* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D007898:Leishmaniasis, Visceral / Q000469:parasitology*; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D010641:Phenotype; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000081406:Peptidyl-Prolyl Isomerase F; D006360:Heat-Shock Proteins; C000631446:PPIF protein, mouse; D015800:Protozoan Proteins; D021983:Cyclophilins
keywords: Chaperone; exosomes; infection; null mutant analysis; proteomics; stress protein; trypanosomatid parasite; virulence