5-Fluorouracil modulates motility and biofilm-associated gene expression in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen in which motility, biofilm formation, and stress adaptation contribute to virulence. Drug repurposing represents a practical strategy for identifying compounds that influence these processes. In this study, the effects of 5-fluorouracil (5-FU) on motility, extracellular DNA (eDNA) production, and gene expression were examined in P. aeruginosa PAO1. Swimming, swarming, and twitching motility assays were performed in the presence of increasing concentrations of 5-FU. Transcriptional responses of motility, rhamnolipid, and DNA repair-associated genes were evaluated using quantitative real-time PCR. eDNA levels were quantified using fluorescence-based assays and visualized by confocal laser scanning microscopy. Exposure to 5-FU resulted in concentration-dependent reductions in swimming, swarming, and twitching motility. These changes were associated with downregulation of multiple genes involved in flagellar and type IV pili function, including motA, flhA, fliD, pilA, and pilI, and reduced expression of lasB and rhlAB. At 24 h, eDNA levels were decreased relative to untreated controls, whereas at 48 h, higher concentrations of 5-FU were associated with increased eDNA accumulation and upregulation of several DNA repair genes, including xthA, nth, recJ, sbcB, and eddB. These results indicate the multifaceted effects of 5-FU on important virulence factors of P. aeruginosa.