PslG-Mediated Disruption of Pseudomonas aeruginosa Biofilms: A Multi-Omics Investigation of Matrix Glycan Release, Metabolic Reprogramming, and Structural Disintegration

Pseudomonas aeruginosa biofilms are recalcitrant to conventional antibiotic therapy due to their extracellular polymeric substance (EPS) matrix, in which Psl polysaccharide serves as the primary structural scaffold. PslG glycoside hydrolase can degrade Psl and trigger biofilm dispersal, yet the dynamic glycan release profile and metabolic consequences remain poorly characterized. Mature P. aeruginosa PAO1 biofilms were treated with PslG (1–1000 nM) for 0–30 min. Biomass was quantified by crystal violet staining. Matrix glycan composition and untargeted metabolomics was performed using LC-MS/MS. Three-dimensional architecture was visualized by confocal laser scanning microscopy (CLSM) with SYTO 9/PI and HHA-FITC staining. PslG induced near-complete loss of Psl-specific fluorescence within 30 min. Counterintuitively, biofilm thickness decreased from 10.8 μm at baseline to approximately 3.0 μm at 5–10 min and partially recovered to 7.2 μm at 30 min, remaining below baseline; the recovery of thickness is progressively due to matrix swelling and bacterial vertical redistribution. PI staining revealed no membrane damage, confirming non-lethal dispersal. Glycomically, galactose, xylose and metabolic intermediates (glucose-6-phosphate) were enriched. Metabolomics revealed significant activation of pentose and glucuronate interconversions, pantothenate and CoA biosynthesis, carbon metabolism, and amino acid biosynthesis. The Psl cleavage mediated by PslG may trigger bacterial dispersal with vertical expansion and metabolic reprogramming toward a planktonic lifestyle. These findings provide mechanistic insights for designing Psl-targeted anti-biofilm therapeutics.