Piperonylpiperazine Targets RhlI to Reduce Virulence and Potentiate EDTA Sensitivity in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a predominant spoilage organism in meat products. The extensive use of antimicrobial preservatives has, however, led to the emergence of resistant strains. Mitigating the resistance of foodborne P. aeruginosa to conventional preservatives has become a critical challenge in food safety. Piperonylpiperazine (Pip), a piperazine derivative sourced from Piper nigrum, was first examined in this work as a novel agent capable of both suppressing virulence and enhancing preservative efficacy against P. aeruginosa, with its mode of action elucidated. At sub-inhibitory concentrations, Pip strongly suppressed the production of virulence factors and potentiated the susceptibility of P. aeruginosa to the common preservative EDTA. Mechanistically, a multi-pronged approach involving pull-down assay, transcriptomic profiling, isothermal titration calorimetry (ITC) analysis, and gene knockout models demonstrated that Pip binds specifically to the LYS-164 and ASP-35 residues of the RhlI synthase, blocking the quorum sensing (QS) signaling cascade. This QS disruption led to reduced virulence factor production and attenuated pathogenicity in a Caenorhabditis elegans model. The compromised QS system subsequently induced oxidative stress, which disrupted cell membrane integrity and permeability, thereby potentiating EDTA's antibacterial action. These findings suggest that Pip is a promising natural additive that can be used in combination with existing preservatives to enhance food safety.