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Subcellular fractionation and molecular characterization of the pellicle and plasmalemma of Neospora caninum

Published online by Cambridge University Press:  16 June 2005

Y. LEI ,
D. BIRCH ,
M. DAVEY  and
J. T. ELLIS
Y. LEI
Affiliation:
Department of Cell and Molecular Biology, Faculty of Science, University of Technology, Sydney, Gore Hill, NSW 2065 Australia
D. BIRCH
Affiliation:
Microscopy Unit, Department of Biological Sciences, University of Macquarie, North Ryde, NSW 2131 Australia
M. DAVEY
Affiliation:
Department of Cell and Molecular Biology, Faculty of Science, University of Technology, Sydney, Gore Hill, NSW 2065 Australia
J. T. ELLIS
Affiliation:
Department of Cell and Molecular Biology, Faculty of Science, University of Technology, Sydney, Gore Hill, NSW 2065 Australia
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Abstract

A characteristic structural feature of Toxoplasma gondii and Neospora caninum is the presence of a triple-membrane pellicle, on the zoite stages of their complex life-cycle. Here we report the results of electron microscopic studies which show that the pellicle is made of a typical plasmalemma covered on its cytoplasmic side by a system of flattened vesicles named the inner membrane complex. Using methods described previously for the purification of pellicle and plasmalemma fractions from T. gondii, we have evaluated the same methodology for the preparation of pellicles and plasmalemma from N. caninum. The approach used involved subcellular fractionation and sucrose gradient centrifugation to prepare fractions containing pellicles. Plasmalemma was prepared by extraction of this fraction with a high salt glycerol treatment. Fractions containing membrane structures were identified by electron microscopy, and the proteins and antigens present in them were subsequently studied by SDS-PAGE and Western blotting. Electron microscopy of the pellicle fractions of N. caninum demonstrated preservation of the triple-membrane structure which is identical to that found in T. gondii. SDS-PAGE of the pellicle fractions revealed it contained several major proteins. Analyses revealed that the plasmalemma of N. caninum contained 2 abundant proteins in addition to other much lower abundance antigens detectable by monoclonal antibodies. These studies therefore report, for the first time, a detailed molecular characterization of the pellicle and plasmalemma of N. caninum.

Keywords

Neospora caninumToxoplasma gondiipellicleplasmalemmasurface antigensubcellular fractionation
Type
Research Article
Information
Parasitology , Volume 131 , Issue 4 , October 2005 , pp. 467 - 475
Copyright
© 2005 Cambridge University Press

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