title: EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall.
abstract: Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.
authors.affiliation: Equipe Parasitologie Moléculaire et Cellulaire, LBP, UMR CNRS 6023, Université Blaise Pascal, 24 Avenue des Landais, 63177 Aubière Cedex, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Peuvel-Fanget
authors.forename: Valérie
authors.initials: V
authors.lastname: Polonais
authors.forename: Damien
authors.initials: D
authors.lastname: Brosson
authors.forename: Catherine
authors.initials: C
authors.lastname: Texier
authors.forename: Lauriane
authors.initials: L
authors.lastname: Kuhn
authors.forename: Pierre
authors.initials: P
authors.lastname: Peyret
authors.forename: Christian
authors.initials: C
authors.lastname: Vivarès
authors.forename: Frédéric
authors.initials: F
authors.lastname: Delbac
mesh_terms: D000595:Amino Acid Sequence; D000908:Antibodies, Fungal / Q000032:analysis; D002473:Cell Wall / Q000737:chemistry; D002686:Chitin; D015825:Chromosomes, Fungal / Q000235:genetics; D003545:Cysteine / Q000032:analysis / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D009670:Encephalitozoon cuniculi / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D016253:Microscopy, Immunoelectron; D012694:Serine / Q000032:analysis / Q000235:genetics; D013172:Spores, Fungal / Q000737:chemistry*
chemical_list: D000908:Antibodies, Fungal; D004270:DNA, Circular; D004271:DNA, Fungal; D005656:Fungal Proteins; D002686:Chitin; D012694:Serine; D003545:Cysteine