Purified Ag43α Protein as a Potential Method for Preventing Escherichia coli Autoaggregation

Abstract

The phenomenon of Escherichia coli autoaggregation and the subsequent biofilm formation poses significant challenges in both healthcare and environmental spheres, primarily due to enhanced bacterial resistance against antibiotics. Autotransporters, particularly Antigen 43 (Ag43), a prominent outer membrane protein in Gram-negative bacteria like E. coli, facilitate bacterial aggregation through their distinct structural domains. Ag43 consists of a secreted passenger domain (Ag43α), an autochaperone domain and a β-barrel domain anchoring the secreted protein to the outer membrane. Previous studies have established the role of the Ag43α subunit in promoting aggregation through self-recognition. Hence, we hypothesise that the application of exogenously purified Ag43α could interrupt these intercellular interactions, thereby inhibiting autoaggregation. We expressed and purified the Ag43α subunit in BL21(DE3) strain of E. coli cells using the pEEKABOO plasmid from Leong et al. to investigate its influence on the aggregation of the Ag43-expressing DH5α E. coli strain. The results exhibited purified Ag43α protein adopting a stable tertiary structure post-translation. Furthermore, preliminary results from a single-trial aggregation assay revealed that introducing 200 µg/mL of purified Ag43α noticeably attenuated autoaggregation in acidic environments. Our preliminary findings provide a basis for further investigation of the Ag43α protein’s potential in modulating E. coli autoaggregation and a possible avenue for the development of novel anti-biofilm therapeutics.