Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collect...

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Bibliographic Details
Published in:International journal for parasitology 2003-07, Vol.33 (7), p.671-679
Main Authors: Hoane, Jessica S., Carruthers, Vernon B., Striepen, Boris, Morrison, David P., Entzeroth, Rolf, Howe, Daniel K.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Apicomplexa
Biological and medical sciences
Blotting, Western
Electrophoresis, Polyacrylamide Gel
Encephalomyelitis, Equine - parasitology
Equine protozoal myeloencephalitis
Fluorescent Antibody Technique
Fluorescent Antibody Technique, Indirect
Fundamental and applied biological sciences. Psychology
Gene Library
Horses
Immune Sera - isolation & purification
Invasion
Life cycle. Host-agent relationship. Pathogenesis
Microneme
Molecular Sequence Data
Neospora
Protozoa
Protozoan Proteins - genetics
Protozoan Proteins - immunology
Protozoan Proteins - isolation & purification
Sarcocystis
Sarcocystis - metabolism
Secretory Vesicles - chemistry
Sequence Alignment
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Toxoplasma
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Summary:Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.
ISSN:0020-7519
1879-0135
DOI:10.1016/S0020-7519(03)00031-6