title: Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan.
abstract: Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.
authors.affiliation: Laboratorio de Estudos Integrados em Bioquimica Microbiana, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941590, Brazil.
authors.forename: Débora L
authors.initials: DL
authors.lastname: Oliveira
authors.forename: Leonardo
authors.initials: L
authors.lastname: Nimrichter
authors.forename: Kildare
authors.initials: K
authors.lastname: Miranda
authors.forename: Susana
authors.initials: S
authors.lastname: Frases
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
authors.forename: Arturo
authors.initials: A
authors.lastname: Casadevall
authors.forename: Marcio L
authors.initials: ML
authors.lastname: Rodrigues
mesh_terms: D001692:Biological Transport; D002458:Cell Fractionation; D002473:Cell Wall / Q000737:chemistry / Q000648:ultrastructure; D016616:Cryoultramicrotomy; D003455:Cryptococcus neoformans / Q000737:chemistry* / Q000648:ultrastructure*; D022162:Cytoplasmic Vesicles / Q000737:chemistry* / Q000648:ultrastructure; D005110:Extracellular Space / Q000737:chemistry; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure; D008563:Membrane Lipids / Q000032:analysis*; D046529:Microscopy, Electron, Transmission; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure; D011134:Polysaccharides / Q000032:analysis*; D037521:Virulence Factors / Q000032:analysis*
chemical_list: D008563:Membrane Lipids; D011134:Polysaccharides; D037521:Virulence Factors; C027478:glucuronoxylomannan