TgRON3 Links the Moving Junction to Microneme Secretion and Parasite Motility in Toxoplasma gondii

Toxoplasma gondii invades host cells through a highly coordinated process centered on the moving junction (MJ). Although core MJ components are essential for efficient invasion, parasites lacking these factors retain limited invasive capacity, suggesting the involvement of additional regulatory elements. To identify such factors, we applied proximity labeling to systematically characterize proteins associated with the MJ component TgRON8. This strategy identified multiple essential proteins, including TgRON3 and TgAPH. Disruption of either gene resulted in a pronounced defect in host cell invasion. Structural modeling and co-immunoprecipitation assays demonstrated that TgRON3 associates with the canonical MJ proteins TgRON2 and TgRON4. In addition, TgRON3 also interacted with two previously uncharacterized transmembrane proteins, TgHTM7 and TgHTM9. Deletion of either TgHTM7 or TgHTM9 led to reduced microneme secretion and substantial transcriptional downregulation of invasion-associated adhesins, including MIC2 and M2AP. Consistent with these molecular defects, parasites lacking TgHTM7 or TgHTM9 exhibited impaired motility, host cell attachment, invasion, and plaque formation. Together, our findings define a regulatory module that links MJ-associated components to microneme secretion and parasite motility, providing new insights into the molecular mechanisms governing T. gondii invasion.