Abstract
Escherichia coli biofilms are a significant source of persistent and chronic infections, with the Antigen 43 (Ag43) protein often helping to mediate their formation through surface self-association, promoting autoaggregation. This study looked to examine whether soluble Ag43α, the passenger domain and functional subunit of Ag43, could function as a competitive inhibitor to block these surface interactions, thereby preventing autoaggregation and biofilm formation. Recombinant Ag43α was expressed in E. coli BL21(DE3) and purified via nickel affinity chromatography. Structural integrity was validated through a limited proteolysis assay, which revealed a trypsin-resistant core, consistent with a folded β-helix. The functional effects of soluble Ag43α were assessed with autoaggregation kinetics and crystal violet staining assays in both fimbriae-deficient (ΔfimA) and fimbriated E. coli backgrounds. Results confirmed that Ag43 expression could drive rapid autoaggregation relative to an empty vector control. While soluble Ag43α did not abolish this phenotype, higher concentrations (100 µg/mL) created a modest and reproducible delay in optical density decline, a proxy for sedimentation. In static biofilm assays, soluble Ag43α did not inhibit biofilm formation in fimbriae-deficient strains. Unexpectedly, in fimbriated strains, Ag43α at low doses (20 µg/mL) significantly enhanced biofilm formation, roughly doubling the intensity in crystal violet staining, while high doses (100 µg/mL) returned biomass to baseline levels. This could possibly be explained by low-dose Ag43α serving as a molecular scaffold in the matrix rather than as a blocker, but at high doses, it ends up saturating binding sites. Furthermore, disruption assays on pre-established biofilms showed no significant reductions across all conditions, indicating that mature Ag43 matrices are resistant to soluble protein interference. These findings ultimately suggest that Ag43 matrices are formed with high avidity interactions, but may be delayed in their formation by the introduction of soluble Ag43α.