Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Koseoglu VK

PROVIDER: S-EPMC6589070 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Köseoğlu Volkan K VK Hall Chelsea P CP Rodríguez-López Eric M EM Agaisse Hervé H

Infection and immunity 20190620 7

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, S1 ...[more]

PMID: 30988059

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Project description:VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF-RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to "fish" out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5'-UUUUaGYcUuUau-3') that is the RNA-converted consensus sequence previously proposed for the VirF-DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF-RNA interaction, in particular when exposed in a stem-loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA-RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem-loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA-RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.

Dataset Information

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

Publications

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

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