Bumped kinase inhibitor BKI-1708 interferes in cytokinesis and drives baryzoite stage conversion in the cyst-forming apicomplexan parasites Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti

ABSTRACT Bumped kinase inhibitors (BKIs) have demonstrated safety and promising efficacy against various apicomplexan pathogens both in vitro and in vivo . However, in the closely related cyst-forming coccidians T. gondii , Neospora caninum and Besnoitia besnoiti , in vitro treatments with a range of BKIs induced the conversion of intracellular tachyzoites into atypical multinucleated complexes (MNCs), also named “baryzoites”. In this study, baryzoites of T. gondii, N. caninum and B. besnoiti generated through exposure of tachyzoites to 2.5 µM BKI-1708 were comparatively assessed. TEM showed that baryzoites contained multiple nuclei, clustered together and separated from the cytoplasmic organelles of newly formed zoites. These zoites do not have outer tachyzoite plasma membrane, were unable to complete cytokinesis, remained intracellular, and were enclosed by a parasitophorous vacuole membrane. TEM demonstrated the presence of an electron-dense cyst wall-like components only in T. gondii baryzoites. Species-specific differences in antigen expression were observed by immunofluorescence using specific antibodies. Comparative proteomic analysis revealed consistent downregulation of ribosomal proteins, proteins associated with secretory organelles, as well as of transcription and translation factors in all baryzoites. Bradyzoite-specific markers were upregulated only in T. gondii baryzoites. In addition, common orthologues of two alveolin-domain filament proteins (IMC7 and IMC12) and a hypothetical protein (TGME49_236950, NCLIV_050850, BESB_060040) were detected at higher abundance in all treated parasites. Overall, baryzoites exhibit distinct phenotypic and proteomic profiles, with ambiguous expression of tachyzoite and bradyzoite antigens, and lacking complete cellular division under drug pressure, suggesting a reversible response to stress rather than progression into a fully differentiated form. Significance Apicomplexan parasites cause serious diseases worldwide, yet treatment options remain limited. A promising group of drugs are BKIs. We investigated how BKI-1708 affects threclosely related T. gondii , N. caninum , and B. besnoiti . Instead of killing the parasites, the drug induced the formation of multinucleated structures termed “baryzoites”. These baryzoites exhibited ambiguous characteristics during the actively growing and dormant stages of the parasite life cycle and were unable to complete normal cell division. Moreover, we observed other key similarities and differences among species including downregulation of ribosomal proteins and transcription/translation factors, while only T. gondii displayed cyst wall formation. Microscopy and proteomics demonstrated that baryzoites represent a distinct stage that is formed upon drug pressure and promotes parasite survival during prolonged drug exposure. These findings highlight the unexpected ways parasites adapt to drug treatment and provide new insights into how BKIs exert their activities.