<i>Toxoplasma</i> effector <i>Tg</i> WIP hijacks dendritic cell actin and motility via Nck1/Grb2 and the WAVE complex

The intracellular parasite <i>Toxoplasma gondii</i> enhances its dissemination to distant organs by hijacking infected leukocytes via a Trojan Horse mechanism. Upon infecting dendritic cells (DCs), <i>Toxoplasma</i> induces a hypermigratory phenotype characterized by podosome dissolution and formation of F-actin stress fibers. We previously showed that these cytoskeletal changes depend on the effector protein <i>Toxoplasma</i> WAVE complex-interacting protein (<i>Tg</i>WIP) secreted from parasites to infected leukocytes. Here, we identify the host adaptor proteins non-catalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck1/2) and growth factor receptor-bound protein 2 (Grb2) as direct <i>Tg</i>WIP interactors. <i>Tg</i>WIP mainly uses two distinct proline-rich regions (PRRs) to interact with Nck1 and Grb2. Mutating these PRRs abrogates <i>Tg</i>WIP binding to Nck1 and Grb2 and diminishes podosome dissolution and DC hypermotility. Furthermore, we show that <i>Tg</i>WIP directly interacts with the actin nucleation-promoting factor WAVE regulatory complex (WRC) via a WRC-interacting receptor sequence (WIRS). Disrupting this interaction also influences actin cytoskeletal remodeling and DC hypermotility. Collectively, our data reveal that <i>Tg</i>WIP directly interacts with multiple actin regulators, including Nck1, Grb2, and the WRC, to remodel the actin cytoskeleton of the host cells, elucidating a key mechanism that <i>Toxoplasma</i> exploits to enhance host cell migration and dissemination.IMPORTANCEThe parasite <i>Toxoplasma gondii</i> spreads throughout the body by hijacking immune cells and boosting their motility. This ability depends on secreted parasite proteins that manipulate the host cell's actin cytoskeleton. One such effector, <i>Toxoplasma gondii</i> WAVE-interacting protein (<i>Tg</i>WIP), induces dramatic changes in host cell shape and movement, but how it does this has remained unclear. Here, we show that <i>Tg</i>WIP directly interacts with multiple host actin-regulatory proteins using distinct sequence motifs. Disrupting these interactions prevents cytoskeletal remodeling and impairs parasite-induced immune cell migration. Our study reveals that <i>Toxoplasma</i> uses defined motifs to co-opt host signaling hubs that control cell motility. Understanding how pathogens exploit the cytoskeleton not only sheds light on host-pathogen interactions but may also reveal broader principles of cell migration relevant to immunity, cancer, and development.