A Newly Discovered Dense Granule Protein 3 in Neospora caninum.

Abstract

Neospora caninum, an obligate intracellular parasite of the phylum Apicomplexa, typically causes an illness known as neosporosis. Dense granule proteins (GRAs) are secreted by apicomplexan and constitute the parasitophorous vacuoles (PVs) structure where tachyzoites proliferate after invasion into host cells. In Toxoplasma gondii, TgGRA3 proteins are strongly associated with PVs membrane and enhance its virulence in vivo, however, research on NcGRA3 has not been reported. Here, a novel NcGRA3 protein in N. caninum was discovered using bioinformatics analysis; the location of NcGRA3 was determined in the extracellular Nc-1 tachyzoites and intracellular PVs after invasion using immunofluorescence assays; the NcGRA3 protein existing form in the PVs membranes was analyzed using western blotting. NcGRA3 shared 41.67% nucleotide homology and 22.17% amino acid homology with TgGRA3. Amino acid sequences ranging from 1 to 25 were signal peptide regions and 135-157 were transmembrane domains. The immunofluorescence assays showed that NcGRA3 was an apical organ secreted dense granule protein and expressed at the posterior end of tachyzoites; the partial co-localization with NcGRA6 in PVs demonstrated that NcGRA3 were expressed in the intravacuolar network structure and PVs membrane. The western blotting assays showed that NcGRA3 protein behaved as an integrated transmembrane protein in PVs. Overall, characterization of the newly discovered NcGRA3 protein will lay a foundation for its function research in the further.