zDHHC20b-mediated palmitoylation of TRIF drives infection-induced necroptosis through the TRIF-RIPK3 axis in teleost monocytes/macrophages

Palmitoylation is a reversible post-translational modification that regulates protein trafficking, stability, and function, yet its role in immune cell death remains poorly defined in teleosts. In this study, using large yellow croaker (Larimichthys crocea) as a teleost model, we identify a critical function for the palmitoyltransferase zDHHC20b (LczDHHC20b) as the infection-responsive zDHHC20 paralog and demonstrated its critical role in promoting necroptosis in monocytes/macrophages (MO/MΦ) during Pseudomonas plecoglossicida infection. Knockdown of LczDHHC20b altered the balance of pro- and anti-inflammatory cytokines and leads to a marked reduction in necroptotic cell death. Mechanistically, acyl-biotin exchange (ABE) assays combined with LC-MS/MS identified TRIF, a key adaptor in innate immune signaling, as a death-related palmitoylation substrate of LczDHHC20b. Palmitoylation of TRIF at Cys92 and Cys142 is progressively enhanced during infection and significantly facilitates its interaction with RIPK3, leading to phosphorylation of RIPK3 and MLKL and the induction of necroptosis. Disruption of this palmitoylation process-via LczDHHC20b knockdown or 2-bromopalmitate treatment-attenuates TRIF-RIPK3 complex formation and suppresses downstream necroptotic signaling. In addition, palmitoylation-deficient TRIF lost its normal association with ER- and TGN-related compartments, suggesting that this modification contributes to the spatial organization of TRIF-dependent signaling. This study provides the first evidence that TRIF undergoes palmitoylation in any species, revealing a previously unrecognized post-translational regulatory mechanism governing TRIF-mediated immune cell death. Together, our findings identify a novel LczDHHC20b-TRIF-RIPK3 regulatory axis controlling necroptosis in teleost macrophages and reveal a previously unrecognized lipid-based mechanism shaping host-pathogen interactions during bacterial infection.