Abstract
Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.
Highlights
Neospora caninum is an obligate intracellular parasite in the phylum Apicomplexa that infects a large number of mammals and causes disease in dogs and cattle [1,2,3]
We chose to collect the fraction positioned just above the rhoptry/dense granule band that contains a mixed fraction of micronemes, rhoptries, dense granules, apicoplasts and mitochondria in T. gondii [24]
Supernatants from hybridoma lines were screened by immunofluorescence assay (IFA) of human foreskin fibroblasts infected with N. caninum
Summary
Neospora caninum is an obligate intracellular parasite in the phylum Apicomplexa that infects a large number of mammals and causes disease in dogs and cattle [1,2,3]. While Toxoplasma infects as much as a third of the human population worldwide and causes severe disease in immunocompromised patients and neonates, Neospora does not appear to infect humans [1,4,6] This key difference in host range of these highly similar parasites emphasizes the importance of comparative analyses of the two organisms, which are currently in progress using genomic, transcriptomic, and proteomic approaches [5,6,7,8]. The dense granules secrete proteins that further modify the vacuole for intracellular survival [13,14,15] Both parasites replicate within similar membrane bound vacuoles and egress from the host cells upon which another lytic cycle is initiated. Intracellular replication is dependent on many processes, but the parasite mitochondrion and apicoplast are key subcellular organelles whose biosynthetic pathways are known targets for therapeutic intervention [16,17,18]
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