IDENTIFICATION AND CHARACTERIZATION OF A DENSE GRANULE-ASSOCIATED PROTEIN IN NEOSPORA-CANINUM TACHYZOITES

Neospora caninum is an apicomplexan parasite which is morphologically and ultrastructurally very similar to Toxoplasma gondii. In order to i dentify molecules involved in host cell entry and subsequent modificat ion of the parasitophorous vacuole, a polyclonal antiserum directed ag ainst N. caninum tachyzoites was raised in a rabbit. Subcellular fract ionation of tachyzoites was performed using the non-ionic detergent Tr iton-X-114. Membrane fractions were analysed by immunoblotting using t he polyclonal antiserum. One of the immunoreactive protein bands had a mol. wt of 33 000 and was subsequently named Nc-p33. Affinity-purifie d anti-Nc-p33 antibodies were used to characterise this polypeptide us ing SDS-PAGE, isoelectric focusing, Western blot analysis and immuno-E M. Nc-p33 was found in two isolates of N. caninum (NC-1 and Liverpool) , but could not be detected in T. gondii tachyzoites. Immunogold EM re vealed that Nc-p33 constituted a dense granule-associated protein, and Western blotting demonstrated that Nc-p33 was most likely identical t o the recently described antigen NCDG1. Shortly after invasion. this d ense granule protein was targeted to the parasitophorous vacuole membr ane, and, at later timepoints after infection, was also found on the p arasitophorous vacuolar network. This suggested that Nc-p33 could play a functional role in the modification of the parasitophorous vacuole and its membrane. (C) 1998 Australian Society for Parasitology. Publis hed by Elsevier Science Ltd.