Brain endothelial spheroids and cortical organoids reveal the impact of Toxoplasma gondii lineage and host-phagocyte-pathogen interactions on colonization.

Toxoplasma gondii chronically infects the central nervous system (CNS), but the mechanisms enabling its traversal of the blood-brain barrier (BBB) remain unclear. Here, we investigated BBB penetration using brain endothelial spheroids and cerebral tissue-derived organoids that recapitulate three-dimensional barrier features. We show that T. gondii tachyzoites efficiently colonize spheroids, without detectable barrier disruption or obligatory parasite replication. Following direct transmigration, tachyzoites invaded and replicated within deeper cell layers. Type I strains (RH, CPS) exhibited higher colonization efficiency than type II strains (PRU, ME49), independent of replication. In contrast, when spheroids were exposed to T. gondii-infected dendritic cells (DCs), both strain types were transported similarly into deep cellular layers. Infected DCs adopted an amoeboid-like migratory phenotype that facilitated parasite transport and subsequent dissemination after egress. Colonization was attenuated by ICAM-1 blockade or heparin treatment, while the parasite effector GRA15, despite modulating DC-endothelial adhesion, did not significantly impact intratissue migration. In contrast, deletion of the effector TgWIP markedly reduced the number of infected DCs entering the spheroids. Similar colonization dynamics were observed in murine cerebral organoids. Collectively, these findings highlight spheroid and organoid models as robust systems for uncovering the cellular and molecular mechanisms underlying T. gondii BBB traversal and CNS colonization.