title: Expression of Pneumocystis jirovecii major surface glycoprotein in Saccharomyces cerevisiae.
abstract: The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine-rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine-rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with β-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg.
authors.affiliation: Critical Care Medicine Department, National Institutes of Health (NIH) Clinical Center, Bethesda, MD 20892, USA.
authors.forename: Geetha
authors.initials: G
authors.lastname: Kutty
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: England
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Kovacs
mesh_terms: D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D015153:Blotting, Western; D005434:Flow Cytometry; D015870:Gene Expression; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified / Q000378:metabolism; D045363:Pneumocystis carinii / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins
keywords: GPI anchored protein; Pneumocystis jirovecii; antigenic variation; major surface glycoprotein; upstream conserved sequence