The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein.
Genes for the Toxoplasma gondii dense granule and rhoptry proteins nucleoside triphosphate hydrolase 3 and ROP2 were expressed at high levels in the closely related parasite N. caninum. The protein products were processed appropriately and were targeted to their correct secretory organelles. NTPase 3 was secreted into the parasitophorous vacuole. The utility of this system was demonstrated in the analysis of a new open reading frame identified upstream of two identical copies of the ROP2 gene. The unknown open reading frame was introduced into Neospora, and transfected parasites were analyzed by immunoblot with antibodies to known T. gondii rhoptry protein families. The transfected Neospora expressed a novel 52 kDa protein, designated ROP8, which localized in the rhoptries. These results illustrate that transfection of known Toxoplasma genes into N. caninum can be used to study their expression, processing and targeting in an immunologically distinct background. They also illustrate the usefulness of N. caninum transfection in the identification and subcellular distribution of proteins encoded by previously uncharacterized Toxoplasma genes.
Beckers
C J
CJ
Yale University School of Medicine, Section of Infectious Diseases, New Haven, CT 06520, USA.
Wakefield
T
T
Joiner
K A
KA
D017766:Acid Anhydride Hydrolases / Q000032:analysis / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000201:enzymology; D005347:Fibroblasts; D018628:Gene Dosage; D015870:Gene Expression; D017125:Genes, Protozoan / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D018688:Neospora / Q000235:genetics*; D043583:Nucleoside-Triphosphatase; D015800:Protozoan Proteins / Q000032:analysis / Q000235:genetics*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000235:genetics*; D014162:Transfection; D014617:Vacuoles / Q000201:enzymology; D014709:Vero Cells
D008565:Membrane Proteins; D015800:Protozoan Proteins; C090555:ROP 2 protein, Toxoplasma gondii; C109324:ROP8 protein, Toxoplasma gondii; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase