Identification and characterization of a new Leishmania major specific 3 0 nucleotidase/nuclease protein Inès Lakhal-Naouar a , Yosser Ben Achour-Chenik a , Yvan Boublik b , Mounira Meddeb c , Ahlem Aamouri c , Abdellatif Fattoum b , Hechmi Louzir b , Mehdi Chenik a, * a LIVGM, Institut Pasteur de Tunis, 13, Place Pasteur, 1002 Tunis-Belvédère, Tunisia b Plateforme de Production des Protéines Recombinantes, CRBM/CNRS, Montpellier, France c Laboratoire d’Histologie et de Cytogénétique Médical, Institut Pasteur de Tunis, 13, Place Pasteur 1002 Tunis-Belvédère, Tunisia article info Article history: Received 18 July 2008 Available online 30 July 2008 Keywords: Leishmania Purine scavenging pathway 3 0 Nucleotidase/nuclease Sandfly Survival abstract We report the characterization of a new Leishmania major gene, lmaj3 0 nt/nu, encoding a 382 amino acids protein, Lmaj3 0 NT/NU, that belongs to the 3 0 nucleotidase/nuclease family. Interestingly, sequence and phylogenetic analysis show that this protein is Leishmania major specific and thus constitutes a new 3 0 nucleotidase/nuclease subgroup. Lmaj3 0 NT/NU displays nuclease enzymatic activity and Western blot analysis shows that it is exclusively expressed in promastigotes. Immunofluorescence microscopy using a specific anti-Lmaj3 0 NT/NU shows that the protein has a plasma membrane localization. Surprisingly, contrary to the previously described Leishmania mexicana 3 0 NT/NU, lmaj3 0 nt/nu is not up-regulated when parasites are cultured under purine starvation conditions. Together, these findings suggest Lmaj3 0 NT/NU may constitute a new important compound of the L. major purine scavenging pathway and could be involved in sandfly parasite survival and colonization. Ó 2008 Elsevier Inc. All rights reserved. The Leishmaniasis are a heterogeneous group of diseases that affects millions of people in tropical and subtropical areas of the world (http://www.who.int/whr/en). Depending on the Leishmania parasite species and on the immunological response of the human host, disease ranges from asymptomatic infections to self-limiting cutaneous lesion(s) or fatal visceral forms. During their life cycle, parasites alternate between flagellated promastigotes in the mid- gut of the sandfly vector and amastigotes in the host macrophage [1]. The ability of Leishmania to survive and grow in different ‘‘hos- tile” environments such as the acidic phagolysosomal vesicles of mammalian macrophages and the midgut of sandflies indicates that it probably evolved adaptative strategies including extracellu- lar nutrients acquisition, release of virulence factors, microbicidal resistance and evasion of host immune responses [2,3]. Leishmania, as other protozoa, are unable of de novo purine bio- synthesis, hence for their survival in host environments, they devel- oped a well organized pathway specialized in extracellular purine salvaging. Acquisition of host purines involves different membrane molecules including specialized purine nucleosides, nucleobase transporters and 3 0 nucleotidase/nuclease (3 0 NT/NU) proteins [4,5]. 3 0 NT/NU is a family of proteins where the active site is exposed on the external face of the surface membrane. It displays two func- tional activities (3 0 nucleotidase and nuclease) and is well conserved in different Leishmania and kinetoplastid species [6–9]. Specific analogies in protein sequence and biochemical and kinetic proper- ties were reported between 3 0 NT/NU and Class I single strand spe- cific nucleases of plants and fungi. Several studies suggest that 3 0 NT/NU has a pivotal role in host purines scavenging by its capacity to generate free nucleosides via the hydrolysis of either exogenous 3 0 nucleotides or nucleic acids. Generated nucleosides are then incorporated into the parasite via specific nucleoside transporters. Here, we report the characterization of a new Leishmania major gene, lmaj3 0 nt/nu, encoding a 382 amino acids protein, Lmaj3 0 NT/ NU, that belongs to the 3 0 NT/NU family. Interestingly, sequence and phylogenetic analysis show that this protein is L. major specific and thus constitutes a new 3 0 NT/NU subgroup. Lmaj3 0 NT/NU is exclusively expressed in promastigotes, displays nuclease enzy- matic activity and has plasma membrane localization. Surprisingly, contrary to the previously described Leishmania mexicana 3 0 NT/NU, lmaj3 0 nt/nu is not up-regulated when parasites are cultured under purine starvation conditions. Together, these findings suggest that Lmaj3 0 NT/NU may constitute a new compound of the L. major pur- ine scavenging pathway that may play a key role in Leishmania– sandfly interaction. Materials and methods Parasites, culture conditions, genomic DNA preparations, and DNA sequencing. One L. major isolate (zymodeme MON-25; MHOM/TN/ 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.07.099 * Corresponding author. Fax: +216 71791833. E-mail address: mehdi.chenik@pasteur.rns.tn (M. Chenik). Biochemical and Biophysical Research Communications 375 (2008) 54–58 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc