Two novel phosphatidylinositol phosphate-binding proteins are identified in Neospora caninum with NcPX1 playing a crucial role.

Neospora caninum is a unicellular parasite that causes neosporosis, a major contributor to reproductive disorders in livestock. Whether phosphatidylinositol phosphate (PIP)-binding proteins play a key role in the life cycle of the parasite is unknown. To investigate the functional roles of PIP-binding proteins in the intracellular parasitism and pathogenicity of N. caninum. We identified two novel PIP-binding proteins, NcPX1 and NcPH1, and evaluated their biological functions using CRISPR/Cas9-generated knockout strains. The Δncpx1 strain displayed reduced replication and invasion, along with altered liposomes accumulation in extracellular parasites and impaired expression of dense granule proteins (GRAs) and microneme proteins (MICs). In vivo assays revealed significantly prolonged survival in mice infected with the Δncpx1 strain, underscoring the role of NcPX1 in pathogenicity. Conversely, the Δncph1 strain showed negligible phenotypic changes. These findings establish NcPX1 as a key determinant of N. caninum infection and highlight PIP-binding proteins as potential therapeutic targets for controlling neosporosis.