Bumped Kinase Inhibitor BKI-1708 Interferes in Cytokinesis and Drives Baryzoite Conversion in the Cyst-Forming Apicomplexan Parasites Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti.

Bumped kinase inhibitors (BKIs) have demonstrated safety and promising efficacy against various apicomplexan pathogens both in vitro and in vivo, but do not act parasiticidal in vitro. In the closely related cyst-forming coccidians Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti, treatments with BKI-1708 induce the conversion of intracellular tachyzoites into atypical multinucleated complexes named "baryzoites". In this study, we comparatively assessed tachyzoites and baryzoites of all three species with respect to ultrastructure, differential antigen expression by immunofluorescence, and overall differential protein expression by MS-proteomics. TEM demonstrated common, but also distinguishing, structural features in baryzoites of the three species. They contained newly formed zoites, unable to complete cytokinesis, and thus they were trapped intracellularly. An electron-dense cyst wall-like structure was found only in T. gondii baryzoites. Species-specific differences in antigen expression were observed by immunofluorescence. Comparative proteomic analysis of baryzoites versus tachyzoites revealed a downregulation of ribosomal proteins, proteins associated with secretory organelles, as well as of transcription and translation factors in baryzoites across all species. Bradyzoite-specific markers were upregulated only in T. gondii baryzoites. Two alveolin-domain filament proteins and a hypothetical protein (TGME49_236950, NCLIV_050850, BESB_060040) were detected at higher abundance in all three species. Thus, baryzoites exhibit distinct phenotypic and proteomic profiles, with ambiguous expression of tachyzoite and bradyzoite antigens, suggesting a reversible response to stress rather than progression into a fully differentiated form.