The Edwardsiella T3SS effector EseJ enhances NF-κB signaling by stabilizing p65 and promoting TAK1 phosphorylation

Edwardsiella piscicida PPD130/91 is an important pathogen that infects both fish and humans. Upon infection, E. piscicida induces strong inflammatory responses through its type III secretion system (T3SS). The underlying mechanisms remain to be defined. Here, we demonstrate that E. piscicida T3SS effector EseJ activates the NF-κB pathway in macrophages, leading to increased transcription and secretion of multiple pro-inflammatory cytokines (e.g., IL-1β and IL-6). EseJ orchestrates NF-κB signaling by interfering with two ubiquitin-dependent mechanisms that are mediated by separate functional domains of EseJ. EseJ binds to the NF-κB subunit p65 via its N-terminal region of amino acids 1-242, preventing the K48-linked polyubiquitination of p65 and its subsequent degradation by the proteasome. This maintains elevated levels of total and phosphorylated p65 (p-p65), facilitating p65 nuclear translocation and enhancing its transcriptional activity. EseJ also interacts with TAK1 and TRAF6 (both are upstream of the NF-κB signaling pathway) via its central domain (aa 243-532), thereby promoting the formation of a stable TRAF6-TAK1 complex. This interaction increases the TRAF6-dependent K27-linked polyubiquitination of TAK1, driving TAK1 phosphorylation and the subsequent activation of the IKK-IκB-NF-κB cascade. Inhibiting TAK1 abrogates this signaling, confirming the essential role of TAK1 in EseJ-induced NF-κB activation. In vivo studies using zebrafish larvae also demonstrated that EseJ is required for E. piscicida to induce the production of pro-inflammatory cytokines. Together, we provide evidence that EseJ targets both TAK1 and p65 to promote NF-κB-dependent inflammatory responses. Given the similarities among EseJ homologues in other bacteria, our finding may reveal a conserved effector-mediated immune signaling across diverse Gram-negative pathogens.