Mucin- and mucus-related phenotypes and in vitro interaction of extraintestinal pathogenic Escherichia coli with human pulmonary cells

Abstract Background Extraintestinal pathogenic Escherichia coli (ExPEC) is a major cause of urinary tract infections (UTI), bacteremia, and meningitis. Although ExPEC have also been associated with pneumonia, the virulence mechanisms that enable colonization and infection remain poorly understood. In the airways, mucus constitutes an important innate defense barrier against bacterial colonization, and its disruption may facilitate access to the epithelial surface. Whether ExPEC possess mucolytic traits that contribute to their interaction with the pulmonary environment remains largely unexplored. Therefore, this study aimed to investigate the mucolytic potential of ExPEC strains and their interaction with pulmonary epithelial cells in vitro. Methods Ten ExPEC strains isolated from bloodstream infections were genotypically and phenotypically characterized for traits potentially relevant to the pulmonary environment. The strains were characterized for phylogroups, serotypes, sequence types, virulence profiles, and the presence of homologs of previously described bacterial mucolytic proteins. Phenotypic assays evaluated the ability to penetrate a mucin barrier, utilize mucin as a carbon source, and disrupt or degrade the mucus produced by Calu-3 human bronchial epithelial cells. Bacterial adherence, cytotoxicity, invasion, and intracellular persistence were also evaluated using A549 alveolar epithelial cells. Results The strains showed diverse phylogenetic backgrounds and virulence gene profiles. Homologs of SslE were detected in nine strains, whereas Vat and Tsh homologs were each identified in three strains. Nine strains penetrated the mucin barrier, and nine showed significantly greater growth in mucin-supplemented minimal medium than in non-supplemented medium. All strains caused some degree of disruption or degradation of the mucus layer produced by Calu-3 cells. Notably, strain EC071 lacked homologs of all mucolytic proteins investigated but efficiently penetrated the mucin barrier and showed high growth when mucin was provided as a carbon source, suggesting the involvement of additional, as yet unidentified factors in this phenotype. In A549 cells, adherence varied among strains, and four strains induced visible cytotoxic effects. Among the highly adherent, non-cytotoxic strains evaluated by transmission electron microscopy, EC028 was observed intracellularly and subsequently showed a marked increase in intracellular bacterial counts, reaching approximately 10 7 CFU/mL after 24 h. Conclusions ExPEC bloodstream isolates display heterogeneous but widespread abilities to interact with mucin, airway mucus, and pulmonary epithelial cells. The lack of a direct correspondence between the mucolytic protein homologs investigated and the observed phenotypes suggests that multiple factors may contribute to mucus interaction and degradation. Together, these mucin- and mucus-related phenotypes, epithelial adherence, cytotoxicity, invasion, and intracellular persistence may represent complementary traits that facilitate ExPEC adaptation to the respiratory environment and potentially contribute to pulmonary infection.