A suite of eight Toxoplasma gondii effectors cooperates to activate the non-canonical NF-κB pathway

ABSTRACT As a master of host-cell reprogramming, Toxoplasma gondii ( T. gondii ) tachyzoites manipulate diverse signaling networks to establish a niche permissive for long-term infection. While the parasite’s subversion of canonical NF-κB signaling (p65/p50) is well established, how infection impacts the non-canonical NF-κB pathway has been largely unexplored. Here, we report that T. gondii infection induces nuclear accumulation of the non-canonical NF-κB subunits RelB and p52 in both human and murine fibroblasts. This response is conserved across both type I and type II parasite genetic backgrounds. We demonstrate that this reprogramming is dependent on the MYR1-mediated export of dense granule effectors. Mechanistically, T. gondii infection drives the depletion of the negative regulator TRAF3, leading to the stabilization of NF-κB-inducing kinase (NIK), phosphorylation of p100, and its subsequent processing into p52. Utilizing a panel of combinatorial knockout parasites, we reveal that no single effector is responsible for this phenotype. Instead, a suite of eight MYR1-dependent effectors, IST, NSM, HCE1/TEEGR, GRA16, GRA18, GRA24, GRA28, and GRA84, functions through a collaborative, additive network to drive most of the non-canonical response. These findings highlight a distributed regulatory strategy used by the parasite to overcome host transcriptional robustness and shape host signaling. IMPORTANCE Toxoplasma gondii infects nearly one-third of the global population and establishes infection by extensively rewiring host immune signaling. While decades of work have focused on how the parasite modulates canonical NF-κB activity, whether it also engages the alternative, non-canonical arm of this pathway has remained unclear. Here, we show that T. gondii tachyzoites activate non-canonical NF-κB signaling, driving nuclear accumulation of RelB/p52 through MYR1-dependent effector export. Unexpectedly, no single effector is responsible. Instead, eight secreted proteins act cooperatively to enable NIK stabilization and engage the non-canonical NF-κB cascade, revealing a networked mode of immune control. This discovery highlights a regulatory logic evolved by the parasite to overcome host transcriptional robustness. Together, these findings identify non-canonical NF-κB activation as a new axis of host-parasite interaction and expand our understanding of how T. gondii reprograms central immune signaling circuits through multi-effector networks.