Serrapeptase Eliminates Escherichia coli Biofilms by Targeting Curli Fibers, Lipopolysaccharides, and Phosphate Metabolism

<i>Escherichia coli</i> biofilms are implicated in the development of persistent infections and increased antibiotic resistance, posing a significant challenge in clinical settings. These biofilms enhance bacterial survival by forming protective extracellular matrices, rendering conventional treatments less effective. Serrapeptase (SPT), a proteolytic enzyme, has emerged as a potential anti-biofilm agent due to its ability to degrade biofilm components and disrupt bacterial adhesion. In this study, we report the inhibitory effect of SPT against <i>E. coli</i> biofilm and its effect on key virulence factors. In vitro assays, including crystal violet staining, optical and fluorescence microscopy, and viability measurements, revealed the dose-dependent inhibition of biofilm formation (IC₅₀ = 14.2 ng/mL), reduced biofilm (-92%, 500 ng/mL) and planktonic viability (-45%, 500 ng/mL), and a marked loss of amyloid curli fibers. SPT treatment also lowered the levels of key virulence factors: cellular and secreted lipopolysaccharides (-76%, 8 ng/mL; -94%, 32 ng/mL), flagellin (-63%, 8 ng/mL), and peptidoglycan (-29%, 125 ng/mL). Mechanistically, SPT induced a phosphate-dysregulating response: secreted alkaline phosphatase activity rose (+70%, 125 ng/mL) while cellular DING/PstS proteins declined (-84%, 64 ng/mL), correlating strongly with biofilm inhibition. In silico docking further suggests direct interactions between SPT and the curli subunits CsgA and CsgB, potentially blocking fiber polymerization. Together, these findings position SPT as a powerful non-antibiotic biofilm disruptor against <i>E. coli</i>, offering a promising strategy to undermine bacterial persistence and resistance by targeting both structural matrix components and metabolic regulatory pathways.