Proteomics-guided insights into the antibiofilm and antivirulence action of 2-hydroxy-4-methoxybenzaldehyde against Listeria monocytogenes.

Listeria monocytogenes is a major foodborne pathogen whose persistence and pathogenicity are largely driven by biofilm formation and virulence factor formation. 2-Hydroxy-4-methoxybenzaldehyde (HMB), a phenolic aldehyde derived from the root of Hemidesmus indicus, exhibits diverse biological activities. However, its antivirulence potential against L. monocytogenes remains unexplored. In this study, we systematically evaluated the antibiofilm and antivirulence potential of HMB against L. monocytogenes and its food-derived isolates. HMB exhibited a minimum inhibitory concentration of 512 μg/mL, while sub-inhibitory concentrations significantly inhibited biofilm formation without affecting planktonic growth. HMB treatment markedly impaired biofilm development on abiotic surfaces and key virulence-associated phenotypes, including cell surface hydrophobicity, listeriolysin, and flagellar motility. Comparative proteomic analysis revealed extensive remodelling of the L. monocytogenes proteome, with 62 downregulated and 43 upregulated proteins, notably affecting pathways related to motility, virulence, and biofilm regulation. These findings were further corroborated by qPCR validation and molecular docking studies, which demonstrated stable interactions between HMB and key virulence-associated proteins, supporting a multi-target mode of action. The in vivo anti-infective potential of HMB was validated using a Galleria mellonella infection model, where HMB treatment exhibited no host toxicity, significantly improved larval survival, and reduced bacterial burden following L. monocytogenes infection. Collectively, this integrated in vitro, proteomic, in silico, and in vivo investigation highlights HMB as a promising natural antivirulence and antibiofilm agent with potential application in mitigating L. monocytogenes contamination and infection in the food safety context.