title: Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene.
abstract: Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO(®) expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD. Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Microbiologie Santé et Environnement, Centre AZM pour la Recherche en Biotechnologie et ses Applications, Université Libanaise, Tripoli, Liban.
authors.forename: Sara
authors.initials: S
authors.lastname: Khalife
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Parasitologie, Faculté de Pharmacie, Univ Lille 2, Lille F-59006, France.
authors.forename: El Moukhtar
authors.initials: el M
authors.lastname: Aliouat
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France.
authors.forename: Nausicaa
authors.initials: N
authors.lastname: Gantois
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Jakobczyk
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France.
authors.forename: François
authors.initials: F
authors.lastname: Demay
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Parasitologie, Faculté de Pharmacie, Univ Lille 2, Lille F-59006, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Chabé
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Parasitologie, Faculté de Pharmacie, Univ Lille 2, Lille F-59006, France.
authors.forename: Muriel
authors.initials: M
authors.lastname: Pottier
authors.affiliation: Laboratoire de Microbiologie Santé et Environnement, Centre AZM pour la Recherche en Biotechnologie et ses Applications, Université Libanaise, Tripoli, Liban.
authors.forename: Fouad
authors.initials: F
authors.lastname: Dabboussi
authors.affiliation: Laboratoire de Microbiologie Santé et Environnement, Centre AZM pour la Recherche en Biotechnologie et ses Applications, Université Libanaise, Tripoli, Liban.
authors.forename: Monzer
authors.initials: M
authors.lastname: Hamze
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Univ Lille Nord de France, Univ Lille 2, Lille F-59045, France.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Dei-Cas
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Parasitologie, Faculté de Pharmacie, Univ Lille 2, Lille F-59006, France. Electronic address: annie.standaert-2@univ-lille2.fr.
authors.forename: Annie
authors.initials: A
authors.lastname: Standaert-Vitse
authors.affiliation: Centre d'Infection et d'Immunité de Lille, INSERM U1019, CNRS UMR 8204, Univ Lille Nord de France, Institut Pasteur de Lille, Univ Lille2, Lille F-59019, France; Laboratoire de Parasitologie, Faculté de Pharmacie, Univ Lille 2, Lille F-59006, France.
authors.forename: Cécile-Marie
authors.initials: CM
authors.lastname: Aliouat-Denis
mesh_terms: D003001:Cloning, Molecular; D015870:Gene Expression; D005816:Genetic Complementation Test; D008928:Mitochondria / Q000201:enzymology*; D045363:Pneumocystis carinii / Q000201:enzymology* / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D013482:Superoxide Dismutase / Q000172:deficiency*
chemical_list: D013482:Superoxide Dismutase
keywords: Antioxidant enzymes; Heterologous expression; Menadione; MnSOD; Oxidative stress; Taphrinomycotina