Dense granule protein 41 of Neospora caninum modulates tachyzoite egress by regulating microneme secretion

Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), secreted by the dense granule, play significant roles in modifying the parasitophorous vacuole, maintenance of morphology, and regulating host-cell interactions. However, their precise involvement in tachyzoite egress remains inadequately characterized. In this study, we identified a homologous gene, Ncgra41, corresponding to the dense granule protein 41 (GRA41) of Toxoplasma gondii, which is associated with egress, utilizing NCBI and ToxoDB databases. NcGRA41 is localized extracellularly within dense granules and intracellularly within parasitic vacuoles. Deletion of NcGRA41 did not affect tachyzoites invasion or proliferation but significantly reduced egress capacity and pathogenicity in mice. The phenotypic characteristics were restored in a complementary strain. Further investigation revealed that the absence of NcGRA41 reduced gliding motility and the transcription level of the subtilisin-like protein (SUB1). A microneme secretion assay demonstrated a significant decrease in NcMIC1 secretion, along with reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings demonstrate that NcGRA41, a novel dense granule protein in N. caninum, modulates tachyzoites egress and influences pathogenicity by regulating microneme secretion.