Role of GRA41 in Neospora caninum pathogenicity: insights into tachyzoite egress and microneme secretion

Background Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), discharged by the dense granule, an essential secretory organelle of Neospora caninum , significantly contribute to the modification of parasitophorous vacuole, maintenance of morphology, and regulation of host cells. However, the precise involvement of these proteins in the egress process of tachyzoites remains inadequately characterized. Methods Comprehensive searches and comparative analyses were conducted of the homologous gene with dense granule protein 41 of Toxoplasma gondii , utilizing the NCBI and ToxoDB databases. Subsequently, we performed online bioinformatics analysis. Additionally, for ascertaining subcellular localization, we created an endogenously labeled strain expressing NcGRA41-3xHA. Employing CRISPR/Cas9 gene editing technology, we constructed the NcGRA41 knockout strain ( Δncgra41 ) and NcGRA41 complementary strain ( comΔncgra41 ) to analyze its phenotypes. To further elucidate the function role of NcGRA41, we performed a micronemes secretion assay and assessed the transcription levels of relevant factors during tachyzoites egress through RT-qPCR. Results NcGRA41 exhibited extracellular localization within dense granules and intracellular distribution in parasitic vacuoles. Deletion of NcGRA41 had no discernible impact on the invasion and proliferation of tachyzoites, but, markedly reducing the capacity for egress and pathogenicity in mice. The complementary strain recovered the phenotypic characteristic of Nc1 parasites. Further investigation revealed that the absence of NcGRA41 led to a reduction in gliding motility and the transcription level of subtilisin-like protein (SUB1). The microneme secretion assay demonstrated a significant decrease in the secretion level of NcMIC1, accompanied by reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings collectively contributed to the ultimate decrease in egress. Conclusions The identification of a novel Neospora caninum dense granule protein, designated NcGRA41, has been achieved. NcGRA41 is implicated in influencing the pathogenicity of N. caninum by modulating tachyzoites egress through the secretion of micronemes.