NcGRA17 is an important regulator of parasitophorous vacuole morphology and pathogenicity of Neospora caninum.

Neospora caninum is an obligate intracellular protozoan parasite that infects a wide range of mammalian species, and particularly causes the reproductive loss in cattle. We identified a novel dense granule protein, N. caninum granule protein 17 (NcGRA17) using the CRISPR/cas9 genome editing system and studied its function. We generated the NcGRA17 knockout strain (ΔNcGRA17) and NcGRA17 complementary strain (iΔNcGRA17). Plaque assays and intracellular proliferation tests showed that the ΔNcGRA17 strain formed smaller plaques and had slower intracellular growth. Mouse virulence assay showed loss of virulence for the ΔNcGRA17 strain. We observed that the parasitophorous vacuoles (PVs) of NcGRA17-deficient parasites have aberrant morphology. To investigate the contribution of NcGRA17 α-helices to aberrant morphology of PVs, we transfected four truncated forms of NcGRA17 into NcGRA17 knockout strain and the phenotypes of these mutants were analysed. Lack of the N-terminal region (NT) failed to target the protein to dense granules, while NcGRA17 (Δα1)-HA, NcGRA17 (Δα2-4)-HA and NcGRA17 (Δα5-8)-HA were targeted to dense granules, but failed to rescue the aberrant PV morphology. Our results indicate that NcGRA17 as a dense granule protein determines PV morphology and pathogenicity, and α-helices of NcGRA17 may be responsible for the aberrant morphology of N. caninum PVs.