Unveiling the impact of cGMP-dependent protein kinase of Neospora caninum on calcium fluxes and egress functions through quantitative phosphoproteome analysis

Neospora caninum, a pathogen associated with abortion in pregnant cattle and motor nerve disorders in dogs, poses a substantial threat. Cyclic GMP-dependent protein kinase (PKG) functions as a central signal transduction hub in apicomplexan parasites. However, PKG has not been characterized in N. caninum, and its targets and pathways controlled by PKG remain unknown. Using a mini auxin-inducible degron system (mAID), we knocked down PKG in N. caninum, demonstrating its indispensable role in tachyzoite invasion and egress from host cells. PKG promotes microneme secretion and enhances tachyzoite gliding motility by elevating intracellular CA²⁺ levels ([CA²⁺]_i). Phosphoproteomics identified 1125 proteins as potential downstream targets of PKG, showing significantly reduced phosphorylation after treatment with the PKG Inhibitor MBP146-78. These proteins are involved in signal transduction, transcriptional regulation, lipid transport and metabolism, vesicle transport, and ion transport. Additionally, CACNAP, a calcium channel-associated protein that facilitates calcium influx at the plasma membrane, plays a supportive role in the egress process of N. caninum. These findings underscore the importance of PKG and its downstream molecules in regulating egress, offering novel insights into the mechanisms underlying the activation of [CA²⁺]_i.