Ag43-Mediated Autoaggregation of Escherichia coli Requires the Passenger and the Autochaperone Domains but In Vitro Passenger Domain Folding and Self-Interaction Does Not Require the Autochaperone Domain

Abstract

Autotransporters, belonging to the Type V secretion system, are a class of proteins found in Gram-negative bacteria, facilitating protein expression across the bacterial outer membrane. Antigen 43 (Ag43), a surface-bound, self-associating autotransporter commonly found in many Escherichia coli strains, plays a role in bacterial autoaggregation and biofilm formation, which are critical for bacterial survival and virulence. Ag43 consists of a signal peptide, a passenger (α) domain, an autochaperone (AC) domain, and a β-barrel translocator domain. While Ag43 self-interaction and its role in autoaggregation are well investigated, the role of each domain remains uncharacterized. In this study, we investigated Ag43 α and AC domains using a multifaceted approach: autoaggregation assays revealed that both domains are essential for Ag43-mediated autoaggregation; trypsin susceptibility assays showed that the α domain alone can maintain structural integrity; and a formaldehyde crosslinking assay captured independent α domain self-interaction in vitro, and suggested a potential role of AC in increasing the efficiency of this process. Altogether, this study characterized Ag43 α and AC domains by looking at their in vitro protein stability and self-interaction, and provided novel insight into Ag43-mediated autoaggregation.