Chain formation mediated by Escherichia coli immunoglobulin-binding proteins EibD and EibG depends on expression levels and localization of proteins.

Escherichia coli expresses immunoglobulin-binding proteins (Eib), a subgroup of trimeric autotransporter adhesins (TAA). Subtypes of Eib proteins mediate unique chain-like adherence patterns and autoaggregation. This study investigates the mechanisms underlying chain formation by EibG and EibD; a chain-forming phenotype of the latter has not been previously described. Using constitutive expression systems, we demonstrate that moderate-level expression of EibG and EibD lead primarily to chain formation, whereas higher expression levels predominantly result in clump formation. Notably, chain and clump formation are not mutually exclusive and can occur simultaneously. Selective deletion of the full head domain, but not the N-terminal domain alone, abolished chain formation, highlighting its critical role. Fluorescence microscopy of mixed cultures showed that chains form through homotypic protein-protein interactions. Investigation revealed EibD and EibG were predominantly localized at cell poles, corresponding to sites of intercellular contact. Functional investigations showed that chain-forming strains exhibited enhanced adhesion to plastic surfaces, a key step in biofilm formation, without affecting autoaggregation. These showed Eib-mediated chain formation depends on protein expression levels, domain architecture, and localization, contributing to bacterial adhesion and potentially pathogenicity. Understanding interactions provides insights into TAA-mediated chain formation and autoaggregation.