Targeting the virulence factor suilysin: a structure-guided discovery of chebulinic acid as a potent antivirulence...

The rapid rise of bacterial resistance has compromised conventional antibiotic efficacy, emphasizing the need for antivirulence-based anti-infective strategies.

Targeting the pore-forming toxin suilysin (SLY), this study aimed to establish a surface plasmon resonance (SPR) screening platform to identify candidates that bind to SLY and to evaluate their antivirulence potential.

A total of 159 plant-derived natural compounds were screened to identify SLY-binding candidates. Functional effects were assessed by hemolysis inhibition, biofilm disruption, and bacterial viability assays. The interaction between chebulinic acid (CA) and SLY was further characterized using molecular docking, molecular dynamics, SPR, and isothermal titration calorimetry (ITC) to determine binding properties, and its impact on ATP synthesis, proton gradient, and genes transcription was analyzed. The enhancement of CA with amoxicillin (AMX) was evaluated in a mouse severe infection model, and safety was assessed by cytotoxicity and hemolysis tests.

As the candidate compound with the strongest anti-hemolysis activity, CA bound SLY with high affinity and markedly suppressed SLY-mediated hemolysis at 0.25 μg/ml, while its minimal inhibitory concentration (MIC) against S. suis was 1024 μg/ml. CA disrupted preformed biofilms, neutralized hemolytic activity in bacterial supernatants, and damaged embedded bacteria. Mechanistically, CA stabilized SLY via multiple hydrogen bonds, restricted its conformational flexibility, impaired ATP synthesis, and downregulated sly, IL-6, TNF-α, and iNOS expression. In vivo, CA combined with AMX markedly improved survival (from 0 to 90%), reduced bacterial burden and tissue lesions, and suppressed systemic inflammation without observable toxicity.

These findings reveal the multifaceted antivirulence mechanism of CA and its enhancement therapeutic potential, offering a promising foundation for developing safe and effective "antivirulence + antibiotic" strategies against multidrug-resistant infections.