Extracellular loops of the β-barrel domain catalyze rapid folding for function of self-associating autotransporters

Bacterial aggregation is a phenotype associated with disease pathogenesis. Aggregate formation enhances biofilm development, host colonization, and resistance to antibiotics and host defenses. Antigen 43 (Ag43) is a surface-located autotransporter produced by pathogenic Escherichia coli that mediates cell aggregation in biofilms. Two Ag43 molecules, each from neighboring bacterial cells, fold into elongated β-helical passenger domains that associate in a head-to-tail manner while being anchored to the cell surface by their outer membrane-embedded β-barrels. In this study, we conduct mutational analyses on Ag43 to show that the β-hairpin structure of the fourth and fifth extracellular loops of the β-barrel domain have a crucial role for passenger domain folding and subsequent formation of bacterial aggregates. This work provides mechanistic insight into the role of the autotransporter β-barrel domain to nucleate the rapid folding of the passenger domain into the β-helix that enables bacterial interactions during infection.