Multi-targeted antimicrobial action of Juglone against Proteus mirabilis biofilm and virulence

Proteus mirabilis is a uropathogen frequently implicated in catheter-associated urinary tract infections (CAUTIs), largely due to its ability to form biofilms and express multiple virulence factors. The limitations of conventional antibiotics, along with increasing resistance rates, necessitate the exploration of natural compounds with broader antimicrobial mechanisms against such pathogens. This study investigated the antibiofilm and anti-virulence potential of Juglone, a phytochemical derived from Juglans regia, against P. mirabilis. A series of in vitro assays demonstrated that Juglone not only inhibited bacterial proliferation in a dose- and time-dependent manner but also significantly restricted biofilm formation, as visualized by FE-SEM, while simultaneously impairing motility and reducing the secretion of critical virulence-associated enzymes. Membrane depolarization and altered membrane fluidity indicated compromised bacterial envelope integrity. Gene expression analysis using RT-qPCR further revealed the downregulation of multiple adherence- and virulence-associated genes. Complementing these findings, molecular docking and molecular dynamics simulations confirmed stable interactions between Juglone and the MrpH adhesin protein, highlighting its potential to interfere with host attachment mechanisms. Importantly, untargeted LC-MS-based metabolomic profiling revealed widespread metabolic reprogramming in Juglone-treated P. mirabilis, characterized by enhanced oxidative stress, redox imbalance, and suppression of core biosynthetic and energy-generating pathways. Taken together, these findings establish Juglone as a promising multi-target antimicrobial agent against P. mirabilis, with potential applications in the prevention of catheter-associated infections.