A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway

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 robust nuclear accumulation of the non-canonical NF-κB subunits RelB and p52 in both human and murine cells. This activation follows a gradual kinetic profile and is conserved across both Type I and Type II parasite genetic backgrounds. We demonstrate that this reprogramming is strictly dependent on the MYR1-dependent 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 trigger the non-canonical response. These findings highlight a distributed regulatory strategy used by the parasite to overcome host transcriptional robustness and shape host signaling.