Construction and biological function of gene knockout strain.

Objectives: Toxoplasmosis is a zoonotic parasitic disease caused by (), which can lead to complications such as encephalitis and ocular toxoplasmosis. The disease becomes more severe when the host's immune system is compromised. Rhoptry proteins are major virulence factors that enable to invade host cells. This study aims to construct a rhoptry protein 41 (/ROP41) gene knockout strain and preliminarily investigate the biological function of .

Methods: Using CRISPR/Cas9 technology, a specific single-guide RNA (sgRNA) for the target gene was designed and linked to a recombinant plasmid. Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes. The recombinant plasmid and the homologous fragments were electroporated into , and PCR identification was performed after drug selection and monoclonal screening. Plaque assays were used to comprehensively assess whether affected the growth and proliferation of in host cells. Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity. The STRING database was utilized to construct a protein-protein interaction (PPI) network, and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.

Results: The gene knockout strain (RH ) was successfully constructed and stably inherited. Plaque assays showed that compared with the parental strain, the number of plaques formed by the gene knockout strain did not significantly decrease, but the reduction in plaque size was statistically significant (<0.05). After the gene was knocked out, the invasion ability of was reduced, but there was no statistically significant difference in its proliferation ability (>0.05). The PPI network revealed that ROP41 was associated with other protein kinases and autophagy-related proteins. Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction, biosynthesis, metabolism, and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.

Conclusions: The RH strain has been successfully constructed. ROP41 primarily affects the ability of to invade host cells and may play a role in signal transduction and autophagy-related pathways between and the host.