Pseudomonas aeruginosa is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. In this study, the synergistic effect of thymoquinone and nisin in reducing biofilm formation of P. aeruginosa on lettuce was evaluated, and their anti-virulence and anti-biofilm mechanisms were also investigated. At concentrations ranging from 0.5 to 2 mg/ml, thymoquinone inhibited the production of autoinducers and virulence factors, and enhanced the susceptibility of P. aeruginosa biofilms to nisin as evidenced by the scanning electron microscopy and confocal laser scanning microscopy. Integrated transcriptomics, metabolomics, and docking analyses indicated that thymoquinone treatment disrupted the quorum sensing (QS) system, altered cell membrane component, and down-regulated the expressions of genes related to virulence, efflux pump, and antioxidation. The changed membrane component and repressed efflux pump system enhanced membrane permeability and facilitated the entrance of nisin into cells, thus improving the susceptibility of biofilms to nisin. The dysfunctional QS and repressed antioxidant enzymes lead to the enhancement of oxidative stress. The enhanced oxidative stress disrupted energy metabolism and protein metabolism and ultimately attenuated the virulence and pathogenicity of P. aeruginosa PAO1. Our study indicated that thymoquinone has the potential to function as a QS-based agent to defend against foodborne pathogens in combination with nisin.
The natural product 4-hydroxycinnamic acid (HA) was firstly isolated from the metabolites of Phomopsis liquidambari, one endophytic fungus from Punica granatum leaves. The anti-QS potential of HA was evaluated by β-galactosidase assay and acylated homoserine lactones (AHL) analysis. The MIC of HA was > 1.20 mM. Exposure to HA at sub-MIC concentrations (0.30–0.60 mM) remarkably reduced the β-galactosidase activity and AHL secretion. Transcriptional analysis by qRT-PCR and docking simulation indicated that HA functions as an anti-QS agent by inhibiting the transcriptional levels of traI and traR rather than signal mimicry. The blocked QS lead to suppressed biofilm formation, motilities, and flagella formation after exposure to HA at concentrations ranging from 0.30 to 0.80 mM. The dysfunctional QS also resulted in repressed antioxidant enzymes and intensified oxidative stress. The intensified oxidative stress destroyed membrane integrity, induced energy supply deficiency, resulted in disorder of protein and nuclear acid metabolism, and ultimately weakened pathogenicity of A. tumefaciens. HA may have promising potential for controlling A. tumefaciens.
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