Authors

Discussion

Babesia divergens is an intraerythrocytic apicomplexan parasite transmitted by Ixodes ricinus ticks and a major cause of bovine babesiosis in Europe, with growing recognition as a severe zoonotic pathogen. Parasite survival and transmission rely on tightly regulated processes, including host cell invasion, intracellular replication, egress, and adaptation to both vertebrate and tick hosts. Calcium-dependent protein kinases (CDPKs) are central regulators of these processes in apicomplexan parasites, yet their individual roles in B. divergens remain poorly understood.

B. divergens genome encodes four distinct BdCDPK isoenzymes. Stage-specific transcriptional profiling in synchronised intraerythrocytic cultures revealed dynamic expression of each bdcdpk associated with developmental transitions, highlighting their potential contributions to parasite biology. BdCDPK4 was recently characterised using inducible knockdown, and the observed phenotype is confirmed here via selective chemical inhibition with bumped kinase inhibitors (BKIs). Inhibition of BdCDPK4 arrested parasite egress without affecting nuclear replication, leading to the accumulation of multinucleated intracellular stages. Comparative phosphoproteomic analysis revealed widespread alterations in phosphorylation patterns, consistent with disruption of a CDPK-centred signalling network. Ultrastructure expansion microscopy confirmed preserved cellular architecture, indicating that the egress block resulted from signalling defects rather than structural damage.

In parallel, conventional knockout of BdCDPK5 demonstrated its essential role in intraerythrocytic replication. Loss of BdCDPK5 function led to complete arrest of parasite reproduction within red blood cells, confirming that BdCDPK5 regulates processes distinct from those controlled by BdCDPK4. To explore functions beyond the blood stage, expression of all four bdcdpks was profiled in I. ricinus tissues following artificial feeding on infected blood. Tissue-specific expression patterns suggest that individual BdCDPKs contribute to parasite differentiation and transmission within the tick vector, supporting stage- and host-specific kinase regulation. Both BdCDPK4 and BdCDPK5 have been recombinantly expressed and are enzymatically active, enabling ongoing biochemical and structural studies, including three-dimensional structure determination, to guide inhibitor development.

Together, these findings establish BdCDPKs as key regulators of B. divergens development across vertebrate and vector hosts. By dissecting the roles of BdCDPK4 and BdCDPK5 and highlighting the broader CDPK signalling network, this work identifies promising targets for interventions aimed at disrupting parasite survival, transmission, and pathogenesis.