Galangin Targets Type IV Pilus to Attenuate Clostridium perfringens Pathogenicity in Broilers Chickens

Clostridium perfringens ( C. perfringens ) induces severe clinical manifestations including necrotic enteritis (NE), enterotoxemia, foodborne illness, and gas gangrene, posing substantial threats to public health and livestock industries. Our study focused on the type IV pilus (TFP) of C. perfringens to elucidate the molecular mechanisms underlying natural compound-mediated bacterial inhibition. Firstly, we identified galangin as a candidate through phenotypic screening of TFP-mediated gliding motility and biofilm formation. Subsequently, the in vitro evaluations encompassed bacterial growth kinetics, minimum inhibitory concentration (MIC) determination, cytotoxicity profiling, and cell adhesion assays. Then, the NE-broilers model was established to assess galangin’s therapeutic potential, incorporating qPCR analysis of virS / virR two-component regulatory genes and TFP-encoding genes ( pilA , pilC , pilD , pilT and pilM ), complemented by transmission electron microscopy (TEM) for TFP ultrastructural characterization. Galangin(8 μg/mL) significantly attenuated bacterial motility (29.79%) and biofilm formation (24.85%). It reduced Caco-2 cell adhesion by 45.72% ( p<0.05 ) while maintaining cellular viability. In vivo administration (20 mg/kg galangin) enhanced broiler average weight (galangin group 99.86 g vs 52.68 g Infection group) and improved feed conversion ratio (3.09 vs 5.76). Histopathological analysis demonstrated reduction in ileal lesion, accompanied by 0.78-log CFU/g bacterial load decrease and normalized inflammatory cytokine levels (IL-1β 38.92%, TNF-α 31.53%, IL-6 31.13%). TEM revealed complete TFP structural disintegration, while qPCR confirmed significant downregulation of virR / virS ( virR :1.89-fold, virS :1.34-fold) and TFP genes ( pilA :1.27-fold, pilD :1.43-fold, pilT :2.11-fold, pilM :1.95-fold). This study establishes galangin as a novel TFP-targeted anti-virulence agent against C. perfringens , providing mechanistic insights and therapeutic validation for NE prevention.