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Identification of a gene cluster for cell-surface genes of the SRS superfamily in Neospora caninum and characterization of the novel SRS9 gene

Published online by Cambridge University Press:  23 September 2011

V. RISCO-CASTILLO ,
V. MARUGÁN-HERNÁNDEZ ,
A. FERNÁNDEZ-GARCÍA ,
A. AGUADO-MARTÍNEZ ,
E. JIMÉNEZ-RUIZ ,
S. RODRÍGUEZ-MARCO ,
G. ÁLVAREZ-GARCÍA  and
L. M. ORTEGA-MORA
V. RISCO-CASTILLO
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
V. MARUGÁN-HERNÁNDEZ
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
A. FERNÁNDEZ-GARCÍA
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
A. AGUADO-MARTÍNEZ
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
E. JIMÉNEZ-RUIZ
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
S. RODRÍGUEZ-MARCO
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
G. ÁLVAREZ-GARCÍA
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
L. M. ORTEGA-MORA*
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
*
*Corresponding author: Tel: +34 913944069. Fax: +34 913944098. E-mail: luis.ortega@vet.ucm.es
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Summary

Here we present the detection of a gene cluster for Neospora caninum surface genes, similar to the Toxoplasma gondii SRS9 locus, and the cloning and characterization of the NcSRS9 gene. PCR genome walking, using NcBSR4 gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to the SRS5 and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtained in vitro and RT-PCR analysis showed no significant variations of NcSRS9 transcripts during in vitro tachyzoite-bradyzoite switch, probably due to incomplete maturity of in vitro bradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding of N. caninum persistence.

Keywords

Neospora caninumbradyzoitegenome walkingSRS9 locusNcSRS9 geneNcSRS9 protein

Information

Type
Research Article
Information
Parasitology , Volume 138 , Issue 14 , December 2011 , pp. 1832 - 1842
Copyright
Copyright © Cambridge University Press 2011

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