Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bacchi CJ, Lane S, Weiss LM, Yarlett N, Takvorian P, Cali A, Wittner M (2001) Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi. J Eukaryot Microbiol 48:374–381
Bacchi CJ, Rattendi D, Faciane E, Yarlett N, Weiss LM, Frydman B, Woster P, Wei B, Marton LJ, Wittner M (2004) Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues. Microbiology 150:1215–1224
Beauvais B, Sarfati C, Challier S, Derouin F (1994) In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi. Antimicrob Agents Chemother 38:2440–2448
Bente M, Harder S, Wiesgigl M, Heukeshoven J, Gelhaus C, Krause E, Clos J, Bruchhaus I (2003) Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani. Proteomics 3:1811–1829
Bigliardi E, Riparbelli MG, Selmi MG, Bini L, Liberatori S, Pallini V, Bernuzzi A, Gatti S, Scaglia M, Sacchi L (1999) Evidence of actin in the cytoskeleton of microsporidia. J Eukaryot Microbiol 46:410–415
Böhne W, Ferguson DJ, Kohler K, Gross U (2000) Developmental expression of a tandemly repeated, glycine- and serine-rich spore wall protein in the microsporidian pathogen Encephalitozoon cuniculi. Infect Immun 68:2268–2275
Brosson D, Kuhn L, Delbac F, Garin J, Vivarès C, Texier C (2006) Proteomic analysis of the eucaryotic parasite Encephalitozoon cuniculi (Microsporidia): a reference map for proteins expressed in late sporogonial stages. Proteomics vol 6 (in press) DOI: 10.1002/pmic.200500796
Chavant P, Taupin V, El Alaoui H, Wawrzyniak I, Chambon C, Prensier G, Méténier G, Vivarès CP (2005) Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme. Int J Parasitol 35:1425–1433
Delbac F, Peyret P, Méténier G, David D, Danchin A, Vivarès CP (1998) On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi. Mol Microbiol 29:825–834
Didier ES (2005) Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop 94:61–76
Fasshauer V, Gross U, Bohne W (2005) The parasitophorous vacuole membrane of Encephalitozoon cuniculi lacks host cell membrane proteins immediately after invasion. Eukaryot Cell 4:221–224
Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419:520–526
Franzen C, Müller A (2001) Microsporidiosis: human diseases and diagnosis. Microbes Infect 3:389–400
Green LC, Didier PJ, Didier ES (1999) Fractionation of sporogonial stages of the microsporidian Encephalitozoon cuniculi by Percoll gradients. J Eukaryot Microbiol 46:434–438
Katinka MD, Duprat S, Cornillot E, Méténier G, Thomarat F, Prensier G, Barbe V, Peyretaillade E, Brottier P, Wincker P, Delbac F, El Alaoui H, Peyret P, Saurin W, Gouy M, Weissenbach J, Vivarès CP (2001) Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 414:450–453
Kima PE, Dunn W (2005) Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles. Microb Pathog 38:139–145
Pakes SP, Shadduck JA, Cali A (1975) Fine structure of Encephalitozoon cuniculi from rabbits, mice and hamsters. J Protozool 22:481–488
Peek R, Delbac F, Speijer D, Polonais V, Greve S, Wentink-Bonnema E, Ringrose J, van Gool T (2005) Carbohydrate moieties of microsporidian polar tube proteins are targeted by immunoglobulin G in immunocompetent individuals. Infect Immun 73:7906–7913
Peuvel I, Peyret P, Méténier G, Vivarès CP, Delbac F (2002) The microsporidian polar tube: evidence for a third polar tube protein (PTP3) in Encephalitozoon cuniculi. Mol Biochem Parasitol 122:69–80
Seleznev KV, Issi IV, Dolgikh VV, Belostotskaya GB, Antova OA, Sokolova JJ (1995) Fractionation of different life cycle stages of microsporidia Nosema grylli from crickets Gryllus bimaculatus by centrifugation in Percoll density gradient for biochemical research. J Eukaryot Microbiol 42:288–292
Taupin V, Méténier G, Delbac F, Vivarès CP, Prensier G (2006) Expression of two cell wall proteins during the intracellular development of Encephalitozoon cuniculi: an immunocytochemical and in situ hybridization study with ultrathin frozen sections. Parasitology Jun 132(Pt 6):815–825 DOI: 10.1017/S0031182005009777
Vavra J, Larson J (1999) Structure of the microsporidia. In: Wittner & Weiss (eds) The microsporidia and microsporidiosis. ASM, Washington, DC, pp 7–84
Xu Y, Takvorian PM, Cali A, Orr G, Weiss LM (2004) Glycosylation of the major polar tube protein of Encephalitozoon hellem, a microsporidian parasite that infects humans. Infect Immun 72:6341–6350
Acknowledgements
We are thankful to Dr. Catherine Texier and Dr. Damien Brosson for providing a copy of their manuscript on spore proteomics (in press) and for helping in the spots determinations of 2-D protein profiles. V. Taupin was supported by a grant from “Ministère de l’Education Nationale, de la Recherche et de la Technologie.”
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Taupin, V., Méténier, G., Vivarès, C.P. et al. An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia). Parasitol Res 99, 708–714 (2006). https://doi.org/10.1007/s00436-006-0231-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-006-0231-y