In silico, transcriptomic and proteomic comparative analysis of Dense Granule Proteins orthologues in Toxoplasma gondii and Neospora caninum

Apicomplexa parasites require a parasitophorous vacuole (PV) for replication and infection maintenance, which is formed through protein secretion from organelles such as dense granules. Thus, Dense granule proteins (GRAs) are important for the intracellular survival of these parasites, as they influence host-parasite interactions, the acquisition of nutrients and the modulation of the host's immune responses. Research has focused on GRAs of Toxoplasma gondii, but little is known about related Neospora caninum. Thus, the aim of this study is to fill this gap by carrying out a bioinformatic, transcriptomic and proteomic analysis, providing information on the conservation and divergence of GRAs in these parasites. For that purpose, we generated a list of predicted GRAs obtained at the Toxoplasma Informatics Resources Database (ToxoDB) and the literature, and checked for N. caninum orthologues, in order to initially analyze the genes for synteny, identity, conserved or unique motifs and to compare their predicted conformational structure. We found a high level of synteny and a reasonable ammino acid similarity. Differences in transmembrane domains and signal peptides indicate possible disparities in subcellular localization and functionality between GRAs, whereas little divergence was found in the presence of other conserved domains and predicted 3D structures. On the other hand, gene expression of the GRAs in T. gondii and N. caninum presented a notable distinction in profile for part of the genes, indicating possible adaptations in the host-parasite interactions. In order to confirm the presence of those proteins in the host-N. caninum interface, we used proximity biotinylation to identify the predicted targets in the PV. We have also generated knockout parasites of selected genes and submitted those to in vitro and in vivo assays to assess their role in host resistance to the infections. In conclusion, we believe our work contributes to a deeper understanding of the molecular aspects, functional diversity and evolutionary history of TgGRAs and NcGRAs, and may have far-reaching implications for the development of interventions and therapies for toxoplasmosis and neosporosis.