Identificação e caracterização funcional de uma nova proteína de grânulos densos em Neospora caninum por meio de edição gênica mediada por CRISPR/Cas9

Abstract

Across apicomplexan parasites, dense granule proteins are key effectors that play essential roles in intracellular parasite survival and host immune modulation, although many remain uncharacterized in Neospora caninum. In this study, we identify and characterize a novel dense granule protein of N. caninum, designated as GRA90, through integrated bioinformatic and experimental approaches. Orthology and synteny analyses demonstrated approximately 50% sequence identity with the corresponding Toxoplasma gondii orthologue, with partial conservation of structural features, indicating evolutionary relatedness with potential functional divergence. CRISPR/Cas9-mediated epitope tagging localized GRA90 to the lumen of the parasitophorous vacuole. Disruption of GRA90 significantly reduced plaque formation, reflecting compromised intracellular replication and/or survival. In a murine model, infection with Δgra90 parasites was associated with greater weight loss and decreased IFN-γ levels during the acute phase, alongside a tendency toward increased parasite burden in the brain during chronic infection. Altogether, these data identify GRA90 as a pro-host effector contributing to parasite fitness and expand current understanding of dense granule protein function in N. caninum.