An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia)

Autor: Christian P. Vivarès, Guy Méténier, Gérard Prensier, Vanessa Taupin
Přispěvatelé: Laboratoire Microorganismes : Génome et Environnement (LMGE), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Centre National de la Recherche Scientifique (CNRS), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Université d'Auvergne - Clermont-Ferrand I (UdA)
Rok vydání: 2006
Předmět:
Density gradient
MESH: Mycology
Mycology
Nosema cuniculi
Cell Line
Microbiology
Fungal Proteins
03 medical and health sciences
Microscopy
Electron
Transmission
MESH: Centrifugation
Density Gradient
MESH: Spores
Fungal
Sporogenesis
Centrifugation
Density Gradient
Animals
Electrophoresis
Gel
Two-Dimensional
Cnidosporidia
MESH: Animals
Centrifugation
Protozoa
Encephalitozoon cuniculi
030304 developmental biology
Gel electrophoresis
0303 health sciences
Fungal protein
Chromatography
General Veterinary
biology
030306 microbiology
General Medicine
Spores
Fungal
MESH: Electrophoresis
Gel
Two-Dimensional
biology.organism_classification
MESH: Cell Line
Spore
Parasite
[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology
Infectious Diseases
Insect Science
Microsporidia
MESH: Microscopy
Electron
Transmission
MESH: Fungal Proteins
Parasitology
MESH: Encephalitozoon cuniculi
Percoll
Zdroj: Parasitology Research / Journal of Parasitology
Parasitology Research / Journal of Parasitology, 2006, 99 (6), pp.708-14. ⟨10.1007/s00436-006-0231-y⟩
ISSN: 1432-1955
0932-0113
DOI: 10.1007/s00436-006-0231-y
Popis: International audience; Intracellular development of microsporidian parasites comprises a proliferative phase (merogony) followed by a differentiation phase (sporogony) leading to the release of resistant spores. Sporogony implies, successively, meront-to-sporont transformation, sporont division into sporoblasts, and sporogenesis. We report a procedure improving the separation of sporogonial stages of Encephalitozoon cuniculi, a species that develops inside parasitophorous vacuoles of mammalian cells. Supernatants of E. cuniculi-infected Madin-Darby canine kidney cell cultures provided a large number of parasites mixed with host-cell debris. This material was gently homogenized in phosphate-buffered saline containing 0.05% saponin and 0.05% Triton X-100 then filtered through glass wool columns. Centrifugation of the filtrate on 70% Percoll-0.23 M sucrose gradient gave a reproducible pattern of bands at different densities. Transmission electron microscopy showed that three of the four collected fractions were free of visible contaminants. Corresponding prominent cell stages were early sporoblasts (fraction B), late sporoblasts plus immature spores (fraction C), and mature spores (fraction D). Further centrifugation of the lightest fraction (A) on 30% Percoll-0.23 M sucrose gradient generated a sporont-rich fraction (A2). First analysis of proteins from fractions A2 and D by two-dimensional gel electrophoresis suggested a potential use of the described method for proteomic profiling.
Databáze: OpenAIRE
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