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

Abstract

<i>Shigella flexneri</i> is an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed that <i>S. flexneri</i> forms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins in <i>S. flexneri</i> biofilm formation. BLAST search analysis revealed that the <i>S. flexneri</i> 2457T genome harbors 4 genes, <i>S1242</i>, <i>S1289</i>, <i>S2406</i>, and <i>icsA</i>, encoding adhesin-like autotransporter proteins. Deletion mutants of the <i>S1242</i>, <i>S1289</i>, <i>S2406</i> and <i>icsA</i> genes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption of <i>icsA</i> abolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required for <i>S. flexneri</i> 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 <i>icsP</i> abrogated IcsA processing in biofilms and enhanced biofilm formation.