4‐Octyl Itaconate Suppresses Virulence of Multidrug‐Resistant Avian Pathogenic Escherichia coli via Cysteine Alkylation of LuxS, Icl, and OmpA

Avian pathogenic Escherichia coli (APEC) is the cause of avian colibacillosis, which leads to huge economic losses in poultry production worldwide, and presents significant zoonotic antimicrobial resistance (AMR) risk. Cell‐permeable itaconate derivatives exhibit dual antibacterial and immunomodulatory properties; however, they have not been systematically evaluated against APEC, and their underlying molecular mechanisms remain poorly defined for this pathogen. In the present study, we systematically explored the anti‐APEC therapeutic potential and mechanism of action of 4‐octyl itaconate (4‐OI) using an integrated approach of in vitro phenotypic, ex vivo macrophage, biochemical genetic, and dual in vivo infection models. In vitro phenotypic assays revealed that 400 μM 4‐OI significantly inhibited the growth of the clinical isolate O78‐type APEC94 and increased its susceptibility to physiological osmotic and acid–base stress (pH 5–7) and decreased the biofilm formation by 64%, swarming motility by 57%, macrophage adhesion by 42%, and cellular invasion by 35%. Ex vivo, 4‐OI showed a dose‐dependent inhibitory effect on the intracellular survival of APEC in RAW264.7 macrophages, with equivalent efficacy to tetracycline, and without any host cell cytotoxicity. Mechanistic qPCR confirmed that 4‐OI inhibits the transcription of five key virulence genes ( fimC , luxS , wza , iroN , and menB ) by 2.4–5.3‐fold. Further biochemical pull‐down, LC‐MS/MS, site‐directed cysteine mutation, and gene knockout/overexpression assays demonstrated that 4‐OI directly alkylates the conserved cysteine residues of LuxS, isocitrate lyase (Icl), and OmpA, thereby simultaneously disrupting bacterial quorum sensing, glyoxylate shunt metabolism, and outer membrane integrity. The virulence‐attenuating effect of 4‐OI was confirmed by metabolite complementation assays, which showed that glyoxylate and menaquinone biosynthesis are defective. In vivo therapeutic evaluation showed that 50 mg/kg 4‐OI could significantly suppress APEC‐induced mouse mortality from 87.5% to 12.5%, reduce bacterial loads in serum and all visceral tissues, and downregulated proinflammatory cytokines (IL‐1β and TNF‐α) to relieve over‐immune pathological damage. 50 mg/kg 4‐OI increased the survival of chicken embryos to 86.7% and reduced the bacterial loads in the allantoic fluids by 83% in poultry‐relevant specific pathogen‐free (SPF) chicken embryo models. Moreover, 4‐OI exhibited antibiofilm and antiadhesion activity against various epidemic APEC serotypes and clinical multidrug‐resistant isolates with no effect on the diversity or composition of chicken cecal microbiota. Collectively, our findings support 4‐OI as a safe alternative to conventional antibiotics for treating avian colibacillosis, acting via cysteine alkylation to repress multiple virulence‐ and metabolic‐related targets. This work establishes a multifaceted preclinical proof‐of‐concept dataset supporting further development of 4‐OI as an anti‐multi‐drug resistance (MDR) candidate agent; additional farm‐relevant in vivo trials will be required prior to real‐world application for safeguarding poultry health and global food safety.