The ecnAB toxin-antitoxin system modulates avian pathogenic Escherichia coli virulence through regulating the capsular sialic acid biosynthesis pathway.

Avian pathogenic Escherichia coli (APEC) causes significant economic losses to the global poultry industry, and its virulence mechanisms remain complex and not fully elucidated. Toxin-antitoxin (TA) systems have recently been implicated in bacterial virulence regulation beyond their traditional roles. This study is the first to reveal the critical role of the ecnAB TA system in APEC pathogenicity. Deletion of ecnAB significantly attenuated bacterial virulence in hosts. Transcriptomic and RT-qPCR analyses demonstrated that the absence of ecnAB led to marked downregulation of the neu gene cluster (including neuB, neuC, neuA, neuS, etc.) responsible for capsular sialic acid synthesis and transport, as well as the kpsM gene. Phenotypic assays confirmed that the ΔecnAB mutant exhibited a sharp reduction in capsular polysaccharide and sialic acid production, accompanied by loss of serum resistance and enhanced clearance by macrophages. Further mechanistic studies revealed that the EcnA antitoxin protein directly binds to the promoter regions of the neu operon and kpsM, positively regulating their transcription. In summary, this study identifies the ecnAB TA system as a novel regulatory hub that directly activates capsular sialic acid biosynthesis, promoting intact capsule formation and conferring critical immune evasion capabilities to APEC. This finding not only deepens the understanding of the functional diversity of TA systems but also provides potential targets for developing novel antimicrobial strategies against APEC infections.