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The virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

ABSTRACT: Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s). Here we confirmed the role of IcsA in cell adhesion and defined the region required for IcsA adhesin activity. Purified IcsA passenger domain was able block S. flexneri adherence and was also used as a molecular probe that recognised multiple components from host cells. The region within IcsA's functional passenger domain (aa 138-148) was identified by mutagenesis. Upon the deletion of this region, the purified IcsA?138-148 was found to no longer block S. flexneri adherence and had reduced ability to interact with host molecules. Furthermore, S. flexneri expressing IcsA?138-148 was found to be significantly defective in both cell adherence and invasion. Taken together, our data identify an adherence region within the IcsA functional domain and provides useful information for designing therapeutics for Shigella infection.

SUBMITTER: Qin J

PROVIDER: S-EPMC6946128 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

Qin Jilong J Doyle Matthew Thomas MT Tran Elizabeth Ngoc Hoa ENH Morona Renato R

PloS one 20200107 1

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s). Here we confirmed the role of IcsA in cell adhesion and defined the region required for IcsA adhesin activity. Purified IcsA passenger domain was able block S. flexneri ...[more]

PMID: 31910229

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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 virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

Publications

The virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

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