Endoplasmic reticulum-associated NcDYRK2 regulates Ca2⁺ dynamics, motility, and development gene expression in Neospora caninum

Neospora caninum is an obligate intracellular apicomplexan parasite and a leading cause of bovine abortion worldwide, imposing substantial economic losses on the cattle industry. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are highly conserved serine/threonine kinases that regulate diverse cellular processes in eukaryotes, including cell cycle control, differentiation, and signal transduction. Despite their established roles in model organisms, the functions of DYRK family members in apicomplexan parasites remain poorly characterized. Given the limited availability of effective chemotherapeutic agents against neosporosis, identifying novel parasite kinases that govern virulence and development may reveal promising drug targets. In this study, we characterized NcDYRK2, a DYRK family kinase in N. caninum. We employed CRISPR-Cas9 gene editing to generate a Δ dyrk2 knockout strain and complemented parasites for phenotypic rescue. Subcellular localization was determined by immunofluorescence assay using epitope-tagged NcDYRK2. The effects of NcDYRK2 on parasite viability were assessed through plaque formation, host cell invasion, intracellular replication, and egress assays. Real-time fluorescence imaging was used to analyze calcium signaling following gene deletion. RNA sequencing was performed to profile transcriptomic changes in Δ dyrk2 parasites relative to wild-type parasites. Virulence was evaluated in a murine infection model, and brain cyst burdens were quantified by qPCR at 40 days post-infection. NcDYRK2 localized predominantly to the endoplasmic reticulum. Deletion of NcDYRK2 significantly enhanced gliding motility and plaque formation, whereas host cell invasion, intracellular replication, and egress remained unaffected. Δ dyrk2 parasites displayed elevated basal intracellular Ca 2 ⁺ concentrations but exhibited a diminished Ca 2 ⁺ response to ionomycin stimulation, indicating disrupted calcium homeostasis. Transcriptomic analysis revealed extensive gene expression alterations upon NcDYRK2 disruption, including downregulation of bradyzoite-associated genes such as BPK1, CST1, MCP3, MCP4, and MAG2, as well as the merozoite-associated gene MIC17A, implicating NcDYRK2 in stage differentiation. Additionally, calmodulin genes CAM1 and CAM3 were transcriptionally upregulated, while PIPLCβ was downregulated. Furthermore, Δ dyrk2 parasites showed markedly attenuated virulence in mice, with significantly reduced brain cyst burdens compared to wild-type controls. Collectively, these findings establish NcDYRK2 as a critical coordinator of Ca 2 ⁺-dependent motility, stage-specific gene expression, and parasite virulence in N. caninum. The endoplasmic reticulum-localized kinase modulates intracellular calcium signaling to regulate gliding behavior and developmental gene programs, while its absence severely compromises in vivo pathogenicity. Given its dispensability for host cell invasion and replication yet essential role in virulence, NcDYRK2 serves as a biologically interesting candidate for further evaluation as a potential target in future anti‑neosporosis research.