The Autotransporter IcsA Promotes Shigella flexneri Biofilm Formation in the Presence of Bile Salts

Shigella flexneri is an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed that S. flexneri forms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins in S. flexneri biofilm formation. BLAST search analysis revealed that the S. flexneri 2457T genome harbors 4 genes, S1242, S1289, S2406, and icsA, encoding adhesin-like autotransporter proteins. Deletion mutants of the S1242, S1289, S2406 and icsA genes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption of icsA abolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required for S. flexneri actin-based motility during intracellular infection. In extracellular biofilms, IcsA was also secreted at the bacterial pole and mediated bacterial cell-cell contacts and aggregative growth in the presence of bile salts. Dissecting individual roles of bile salts showed that deoxycholate is a robust biofilm inducer compared to cholate. The release of the extracellular domain of IcsA through IcsP-mediated cleavage was greater in the presence of cholate, suggesting that the robustness of biofilm formation was inversely correlated with IcsA processing. Accordingly, deletion of icsP abrogated IcsA processing in biofilms and enhanced biofilm formation.