title: Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans.
abstract: Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence.
authors.affiliation: Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan.
authors.forename: Kiminori
authors.initials: K
authors.lastname: Shimizu
authors.affiliation: Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan; Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, Mutsuura-higashi 1-50-1, Kanazawa-ku, Yokohama 236-8501, Japan. Electronic address: shimizuy@kanto-gakuin.ac.jp.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Imanishi
authors.affiliation: Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Toh-e
authors.affiliation: Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Uno
authors.affiliation: Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan.
authors.forename: Hiroji
authors.initials: H
authors.lastname: Chibana
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, WI 53706, USA; Department of Medical Microbiology & Immunology, School of Medicine and Public Health, University of Wisconsin-Madison, WI 53706, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Hull
authors.affiliation: Medical Mycology Research Center, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8673, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Kawamoto
mesh_terms: D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000235:genetics; D003979:Diazonium Compounds / Q000378:metabolism; D055786:Gene Knockout Techniques; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D013194:Staining and Labeling
chemical_list: D003979:Diazonium Compounds; C020747:Fast Blue B; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase
keywords: Agrobacterium tumefaciens-mediated transformation; Cryptococcus gattii; Cryptococcus neoformans; Diazonium blue B; Glycosylation