The inhibition effect and mechanism of litsea cubeba essential oil on the hemolytic activity of listeriolysin O

This study investigated the use of Litsea cubeba essential oil (LC-EO) as an anti-virulence agent targeting listeriolysin O (LLO) of Listeria monocytogenes (L. monocytogenes). The hemolysis assay showed a significant decrease in the hemolytic activity of LLO by 90.7% and 70.2% after exposure to 500 mu g mL-1 LC-EO or 700 mu g mL-1 citral (the main component of LC-EO), respectively. This suggested that citral plays a major role in the anti-virulence function against LLO. The experiments including UV-visible absorption spectroscopy, fluorescence quenching and three-dimensional fluorescence spectroscopy demonstrated that exposure to citral leads to significant changes in the conformation of LLO, potentially resulting in the unfolding of its structure and the formation of a ground-state complex. Synchronous fluorescence spectra analysis indicated that the binding site of citral could be the undecapeptide region of LLO. Additionally, the results of molecular docking and the acid-base effect experiment of different aldehydes and phenols revealed that the cysteine residue of LLO and the alpha, beta-unsaturated aldehyde of citral are likely to form a covalent bond through Michael addition. This interaction is believed to be the primary reason for the loss of hemolytic activity. As a result, this work provides a new approach for the development of LLO-targeting anti-virulence agents and holds promise for expanding the application of LC-EO in the field of food microbiological safety.