GRA12, a novel dense granule protein from Neospora caninum |
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| Institution: | 1. Key Laboratory of Zoonosis Research by Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;2. Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun 130062, Jilin, China;3. Laboratory of Veterinary Microbiology, Department of Veterinary Medicine, Yanbian University, Yanji 133002, Jilin, China;1. Department of Parasitology, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;2. National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;1. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Rivadavia 1917, C1033AAJ Buenos Aires, Argentina;2. Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria Balcarce, Ruta 226 Km 73.5, 7620 Balcarce, Buenos Aires, Argentina;3. Centro de Investigación en Abejas Sociales, Departamento de Biología, Facultad de Ciencias Exactas y Naturales, UNMDP, Funes 3350, 7600 Mar del Plata, Buenos Aires, Argentina;4. Centro de Investigación Veterinaria de Tandil (CIVETAN, CONICET-CICPBA), Facultad de Ciencias Veterinarias, UNCPBA, Paraje Arroyo Seco s/n, 7000 Tandil, Buenos Aires, Argentina;5. Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Canada |
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| Abstract: | Neospora caninum, an obligate intracellular parasite of the phylum Apicomplexa, is a major cause of abortion in cattle. After invasion, tachyzoites can reside in the parasitophorous vacuole (PV) and ingest nutrition through the intravacuolar network (IVN). Secreted dense granule proteins of N. caninum (NcGRAs) may play important roles in maintaining the structures of the PV and IVN. In this study, we predicted a NcGRA12 gene; aligned it with Toxoplasma gondii GRA12 for homology analysis; and analyzed the ORF, signal peptide and transmembrane domain. Then, we cloned the NcGRA12 gene, expressed the NcGRA12 protein, prepared polyclonal antibodies, and carried out colocalization analysis of NcGRA12 with NcGRA6 in extracellular tachyzoites and intracellular PVs using an immunofluorescence assay (IFA). Finally, we determined the solubility of the NcGRA12 protein. The results showed that NcGRA12 shared 59.13% nucleotide homology and 44.9% amino acid homology with TgGRA12. There was no predicted signal peptide or transmembrane domain. IFA data of extracellular tachyzoites showed that the NcGRA12 protein was secreted by the apical organ and located at the posterior end of tachyzoites, which was consistent with TgGRA12. IFA data of intracellular PVs identified NcGRA12 in the IVN membranes. Moreover, NcGRA12 could colocalize with NcGRA6 in intracellular PVs but not extracellular tachyzoites. Solubility analysis showed that NcGRA12 existed in soluble and membrane-related forms in the PV. Overall, we provide the first report of the novel NcGRA12 protein and verify that it is associated with the IVN membranes of PVs in N. caninum. These data lay a foundation for further research into the function of NcGRA12. |
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