Abstract
Autoaggregation by Escherichia coli promotes the formation of persistent biofilms that can cause chronic bacterial infections. Antigen 43 is a type V autotransporter that drives autoaggregation through self-association. Antigen 43 variants are grouped into six phylogenetic classes and display diverse capacities to form homotypic and heterotypic interactions during autoaggregation. Previous work proposed that soluble antigen 43 of the C1 class has therapeutic potential by competitive inhibition of antigen 43-mediated autoaggregation. Compared to variants of the C1 class, surface-displayed variants of the C4 class engage in broader heterotypic interactions with other antigen 43 classes. On this basis, we hypothesized that soluble C4 antigen 43 would display comparable inhibition of autoaggregation to soluble C1 antigen 43 but have greater therapeutic potential. To investigate this, we constructed a plasmid which expresses the passenger domain from the C4 class of antigen 43 by Gibson Assembly. We induced protein production from the plasmid using isopropyl β-D-1-thiogalactopyranoside and purified it by nickel resin chromatography. We then assessed autoaggregation in a BW25113 pBAD-Ag43 model at pH 7.4 to determine the capacity of the soluble passenger domain of antigen 43 of the C4 class to inhibit antigen 43-mediated autoaggregation and compared it to the C1 class. Technical replicates show that neither of the proteins significantly inhibit E. coli autoaggregation at the concentrations tested. This study provides a model through which E. coli autoaggregation by antigen 43 C1, C4, and other phylogenetic classes can be studied.