Acacetin Alleviates Listeria monocytogenes Virulence Both In Vitro and In Vivo via the Inhibition of Listeriolysin O.

Abstract

Listeria monocytogenes is a ubiquitous Gram-positive foodborne pathogen that is responsible for listeriosis in both humans and several animal species. The bacterium secretes a pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), a major virulence factor involved in the activation of cellular processes. The ability of LLO to lyse erythrocytes is a measure of LLO activity. We used hemolytic activity assay to screen the LLO inhibitors. Acacetin was found to be an LLO inhibitor, which is a di-hydroxy and mono-methoxy flavone present in various plants, including Black locust, Damiana, and Silver birch. As the features of acacetin are of low toxicity and have less acquired resistance, it comes to a hotspot in drug development. In our study, we report that acacetin antagonized the hemolytic activity of L. monocytogenes culture supernatants and purified LLO by directly interfering with the formation of oligomers without inhibiting the bacterial growth and the expression of LLO. Acacetin also relieved the injury of alveolar epithelial cells by inhibiting LLO activity. Further, acacetin significantly promoted the clearance of L. monocytogenes and alleviated the histopathological damage, thereby raising survival rate, which conferred mice with effective protection against L. monocytogenes infection. Using molecular docking and dynamics simulation, we further proved the mechanism of acacetin antagonizing LLO pore-forming activity by direct binding to the second membrane-inserting helix bundle (HB2) of LLO domain 3. These data suggested that acacetin recedes the virulence of L. monocytogenes both in vivo and in vitro, and this study provided a promising candidate and potential alternative for the prevention and treatment of L. monocytogenes infections.