Identification of Novel Proteins in Neospora caninum Using an Organelle Purification and Monoclonal Antibody Approach

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.

Production, Characterization and Applications for Toxoplasma gondii-Specific Polyclonal Chicken Egg Yolk Immunoglobulins

“…In that sense, immunizations using single antigens of T. gondii could be employed to better understand the role of the selected molecules, as tools to improve the diagnosis of acute toxoplasmosis [33] or the knowledge of hostparasite interaction mechanisms [34]. Additionally, the identification of conserved surface antigens among apicomplexan organisms is usually performed by mammal specific IgG antibodies and contributes to our understanding of parasite evolution [35][36][37].…”

The molecular basis for the distinct host and tissue tropisms of coccidian parasites

“…Microneme discharge deploys host adhesive proteins (MICs) onto the parasite surface, which bind to glycan receptors on the host cell surface, and establish a strong initial attachment [14]. This is promptly followed by secretion of rhoptry proteins [15], insertion of which into the host plasma membrane provides a receptor for the conserved microneme protein, AMA1 [16] giving rise to the 'moving junction'; a tight association between host and parasite membranes [17][18][19][20]. Powered by the glideosome, the moving junction is translocated across the parasite surface toward the posterior pole whilst MIC proteins are shed from the junction via rhomboid protease activity [21,22], forcing engulfment of the parasite by the host cell.…”

Importance of serological cross-reactivity amongToxoplasma gondii, Hammondiaspp.,Neosporaspp.,Sarcocystisspp. andBesnoitia besnoiti

“…Liao et al (2005 a ) produced 384 MAbs against N. caninum by immunizing mice with N. caninum tachyzoites; 10 of the 384 MAbs were also reactive against T. gondii tachyzoites. Similarly, Sohn et al (2011) developed 46 MAbs using a mouse immunized with a mixed fraction of N. caninum organelles and some of the MAbs cross-reacted with T. gondii . MAbs generated to oocyst antigens of T. gondii cross-reacted by immunofluorescence with the sporocyst wall (Dumetre and Darde, 2007) and tissue the cyst wall of N. caninum (Gondim et al 2016).…”