-Octyl Itaconate Attenuates Avian Pathogenic Escherichia coli (APEC) Pathogenicity via Virulence Gene Repression, Metabolic Disruption, and Modulation of Host Inflammatory Responses

Avian pathogenic Escherichia coli (APEC) is a leading cause of avian colibacillosis, inflicting substantial economic losses on the global poultry industry and posing zoonotic risks due to rising antimicrobial resistance (AMR). Itaconate and its derivatives have emerged as promising antimicrobial and immunomodulatory agents, but their efficacy against APEC remains unexplored. Here, we systematically investigated the anti-APEC potential of 4-octyl itaconate (4-OI), a cell-permeable itaconate derivative, using in vitro, ex vivo, and in vivo models. In vitro assays demonstrated that 4-OI (400 μM) impaired APEC94 (O78 serotype) growth, enhanced susceptibility to osmotic/acid-base stress (pH 5–7), and reduced biofilm formation (64%), swarming motility (57%), host cell adhesion (42%), and invasion (35%). Ex vivo, 4-OI suppressed intracellular APEC94 survival in RAW 264.7 macrophages in a dose-dependent manner compared to tetracycline without inducing host cell cytotoxicity. Mechanistically, qPCR validated that 4-OI repressed key virulence genes (fimC, luxS, iroN, wza, menB) with fold repression ranging from 2.4× to 5.3×, linking phenotypic effects to transcriptional regulation. In vivo, 4-OI (50 mg/kg) reduced APEC94-induced mortality in mice from 87.5% to 12.5%, lowered bacterial loads in systemic/mucosal tissues, and downregulated pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) to mitigate immunopathology. In a poultry-relevant chicken embryo model, 50 mg/kg 4-OI increased survival to 86.7% and reduced allantoic fluid bacterial loads by 83%. Collectively, our findings demonstrate that 4-OI targets APEC’s core pathogenic traits by disrupting metabolism and repressing virulence genes, offering a safe, effective alternative to conventional antibiotics for the treatment of pathogenic Escherichia coli infections. This study provides a translational framework for developing 4-OI-based interventions to combat AMR and safeguard poultry health.