Molecular & Biochemical Parasitology 137 (2004) 121–132 Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum  Natasha Sienkiewicz a,1 , Wassim Daher b,1 , Daniel Dive b , Carsten Wrenger a , Eric Viscogliosi b , René Wintjens c , Helène Jouin b,d , Monique Capron b , Sylke Müller a,∗ , Jamal Khalife b,2 a Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, WTB/MSI Complex, Dundee DD15EH, UK b Unité Inserm 547/IPL, Institut Pasteur, 1 rue du Pr Calmette, B.P. 245, F-59019 Lille Cedex, France c Université Libre de Bruxelles, Institut de Pharmacie, Chimie Générale, CP 206/04, Campus de la Plaine, Bld du Triomphe, B-1050 Bruxelles, Belgium d Immunologie Moléculaire des Parasites, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Received 26 February 2004; received in revised form 12 May 2004; accepted 15 May 2004 Available online 19 June 2004 Abstract The intraerythrocytic stages of Plasmodium falciparum are exposed to oxidative stress and require functional anti-oxidant systems to survive. In addition to the parasite’s known iron-dependent superoxide dismutase PfSOD1, a second SOD gene (PfSOD2) interrupted by 8 introns was identified on chromosome 6. Molecular modelling shows that the structure of PfSOD2 is similar to other iron-dependent SODs and phylogenetic analysis suggests PfSOD1 and PfSOD2 are the result of an ancestral gene duplication. The deduced amino acid sequence of PfSOD2 is similar to PfSOD1 but has a long N-terminal extension. Immunofluorescence studies show that PfSOD1 is cytosolic, whereas the N-terminal extension of PfSOD2 targets a green fluorescent protein fusion into the parasite’s mitochondrion. Both SOD genes are transcribed during the erythrocytic cycle with PfSOD1 mRNA levels up to 35-fold higher than those of PfSOD2. Northern blots demonstrated that the mRNA levels of both SOD genes are up-regulated upon exposure to oxidative stress. © 2004 Elsevier B.V. All rights reserved. Keywords: Superoxide dismutase; Mitochondrion; Transfection; Quantitative RT-PCR; Malaria; Antioxidant; Oxidative stress 1. Introduction Superoxide dismutases (SODs) are metallo-proteins that occur ubiquitously in nature and catalyse the dismutation of superoxide anions to form molecular oxygen and hydrogen peroxide following the reactions shown as follows: Me ox + O 2 •- → Me red + O 2 Me red + O 2 •- + 2H + → Me ox + H 2 O 2 Abbreviations: GFP, green fluorescent protein; ORF, open reading frame; PfSOD1, superoxide dismutase 1 of Plasmodium falciparum; Pf- SOD2, superoxide dismutase 2 of P. falciparum; Q-PCR, quantitative polymerase chain reaction; RT, reverse transcriptase; RT-PCR, reverse transcriptase polymerase chain reaction; SOD, superoxide dismutase  Note: Genbank accession number for PfSOD2: AY586514. ∗ Corresponding author. Tel.: +44 1382 345760; fax: +44 1382 345764. E-mail addresses: s.muller@dundee.ac.uk (S. Müller), jamal.khalife@pasteur-lille.fr (J. Khalife). 1 These authors contributed equally to this work. 2 Co-corresponding author. Tel.: +33 3208 77968; fax: +33 3208 77888. SODs are assigned into distinct families, based on their metal cofactors Cu/Zn, Fe or Mn. Mammalian cells contain cytosolic Cu/ZnSODs and MnSODs in their mitochondria [1]. Eubacteria generally rely on Mn and FeSODs which have very similar primary, secondary and tertiary structures but have a strict selectivity for their metal co-factor; they are evolutionarily unrelated to Cu/ZnSODs [1]. Another group of SODs which uses either Fe or Mn as their metal co-factor are named cambialistic SODs and have been identified in a variety of bacteria [2,3]. The sequencing of the Plasmodium falciparum genome allowed large scale analyses of the parasite’s transcriptome with the aim of identifying critical genes for parasite devel- opment and survival. Serial analyses of gene expression and DNA microarray experiments described the major metabolic pathways and the profile of developmentally-regulated genes in blood-stage parasites under normal culture con- ditions [4,5]. In addition, the data currently available are exploitable for comparative analyses with other organisms and known genes. This provides valuable information on the Plasmodium genome and biology and improves the abil- 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.05.005