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Stretching fibronectin fibres disrupts binding of bacterial adhesins by physically destroying an epitope.

Chabria M, Hertig S, Smith ML, Vogel V - Nat Commun (2010)

Bottom Line: Heparin reduces binding but does not eliminate mechanosensitivity.The mechanical switch described here operates differently from the catch bond mechanism that Escherichia coli uses to adhere to surfaces under fluid flow.Demonstrating the existence of a mechanosensitive cell-binding site provides a new perspective on how the mechanobiology of ECM might regulate bacterial and cell-binding events, virulence and the course of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials, ETH Zurich, Zürich CH-8093, Switzerland.

ABSTRACT
Although soluble inhibitors are frequently used to block cell binding to the extracellular matrix (ECM), mechanical stretching of a protein fibre alone can physically destroy a cell-binding site. Here, we show using binding assays and steered molecular dynamics that mechanical tension along fibronectin (Fn) fibres causes a structural mismatch between Fn-binding proteins from Streptococcus dysgalactiae and Staphylococcus aureus. Both adhesins target a multimodular site on Fn that is switched to low affinity by stretching the intermodular distances on Fn. Heparin reduces binding but does not eliminate mechanosensitivity. These adhesins might thus preferentially bind to sites at which ECM fibres are cleaved, such as wounds or inflamed tissues. The mechanical switch described here operates differently from the catch bond mechanism that Escherichia coli uses to adhere to surfaces under fluid flow. Demonstrating the existence of a mechanosensitive cell-binding site provides a new perspective on how the mechanobiology of ECM might regulate bacterial and cell-binding events, virulence and the course of infection.

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Fn fibre stretch assay to quantify mechanosensitive bacterial peptide binding.(a) Fn fibres were made by dipping a sharp tip into a drop of PBS containing 0.5 g l−1 Fn (5% Fn-Cy5 and 95% unlabelled Fn), which is then gently pulled back to induce Fn polymerization into fibres. (b) These prestrained fibres are subsequently deposited onto stretchable silicone sheets27 in various orientations to the horizontal strain axis, resulting in variable degrees of fibre strains. (c) Confocal micrograph depicting fluorescent intensity ratios of bacterial peptide B3C-488 binding to Fn-Cy5 fibres, shown here in false colours. Scale bar: 50 μm.
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f2: Fn fibre stretch assay to quantify mechanosensitive bacterial peptide binding.(a) Fn fibres were made by dipping a sharp tip into a drop of PBS containing 0.5 g l−1 Fn (5% Fn-Cy5 and 95% unlabelled Fn), which is then gently pulled back to induce Fn polymerization into fibres. (b) These prestrained fibres are subsequently deposited onto stretchable silicone sheets27 in various orientations to the horizontal strain axis, resulting in variable degrees of fibre strains. (c) Confocal micrograph depicting fluorescent intensity ratios of bacterial peptide B3C-488 binding to Fn-Cy5 fibres, shown here in false colours. Scale bar: 50 μm.

Mentions: To determine whether mechanical strain alters the binding of a bacterial adhesin, a fragment B3 of FnBR-4 from S. dysgalactiae (Fig. 1b) was used. The B3 peptide (Fig. 1c) was synthesized with an additional N-terminal cysteine residue (B3C) in order to label it with Alexa Flour-488 dye (B3C-488). A binding assay that allows the stretching of single Fn fibres through the full range of physiologically relevant conformations (from fully relaxed to breakage)27 was used in combination with optical colocalization studies to experimentally verify strain-dependent binding25. Fn fibres were manually deposited on stretchable silicone sheets (Fig. 2a,b). To quantify the binding, ratiometric measurements of labelled B3C-488 bound to Fn fibres that contained Cy5-labelled Fn were observed as a function of fibre extension (mechanical strain; Fig. 2c). Depositing fibres in different orientations on the same sheet allowed an overview of differential binding as a function of strain. To increase statistical significance, we deposited fibres parallel to each other that are under the same mechanical strain (Fig. 3a–f). The intensity ratio of the labelled B3C-488 (IB3C-488) per labelled Fn (IFn-Cy5) versus the mechanical strain shows that the binding of the bacterial peptide B3C-488 decreased significantly when fibres were stretched (Fig. 4a).


Stretching fibronectin fibres disrupts binding of bacterial adhesins by physically destroying an epitope.

Chabria M, Hertig S, Smith ML, Vogel V - Nat Commun (2010)

Fn fibre stretch assay to quantify mechanosensitive bacterial peptide binding.(a) Fn fibres were made by dipping a sharp tip into a drop of PBS containing 0.5 g l−1 Fn (5% Fn-Cy5 and 95% unlabelled Fn), which is then gently pulled back to induce Fn polymerization into fibres. (b) These prestrained fibres are subsequently deposited onto stretchable silicone sheets27 in various orientations to the horizontal strain axis, resulting in variable degrees of fibre strains. (c) Confocal micrograph depicting fluorescent intensity ratios of bacterial peptide B3C-488 binding to Fn-Cy5 fibres, shown here in false colours. Scale bar: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105298&req=5

f2: Fn fibre stretch assay to quantify mechanosensitive bacterial peptide binding.(a) Fn fibres were made by dipping a sharp tip into a drop of PBS containing 0.5 g l−1 Fn (5% Fn-Cy5 and 95% unlabelled Fn), which is then gently pulled back to induce Fn polymerization into fibres. (b) These prestrained fibres are subsequently deposited onto stretchable silicone sheets27 in various orientations to the horizontal strain axis, resulting in variable degrees of fibre strains. (c) Confocal micrograph depicting fluorescent intensity ratios of bacterial peptide B3C-488 binding to Fn-Cy5 fibres, shown here in false colours. Scale bar: 50 μm.
Mentions: To determine whether mechanical strain alters the binding of a bacterial adhesin, a fragment B3 of FnBR-4 from S. dysgalactiae (Fig. 1b) was used. The B3 peptide (Fig. 1c) was synthesized with an additional N-terminal cysteine residue (B3C) in order to label it with Alexa Flour-488 dye (B3C-488). A binding assay that allows the stretching of single Fn fibres through the full range of physiologically relevant conformations (from fully relaxed to breakage)27 was used in combination with optical colocalization studies to experimentally verify strain-dependent binding25. Fn fibres were manually deposited on stretchable silicone sheets (Fig. 2a,b). To quantify the binding, ratiometric measurements of labelled B3C-488 bound to Fn fibres that contained Cy5-labelled Fn were observed as a function of fibre extension (mechanical strain; Fig. 2c). Depositing fibres in different orientations on the same sheet allowed an overview of differential binding as a function of strain. To increase statistical significance, we deposited fibres parallel to each other that are under the same mechanical strain (Fig. 3a–f). The intensity ratio of the labelled B3C-488 (IB3C-488) per labelled Fn (IFn-Cy5) versus the mechanical strain shows that the binding of the bacterial peptide B3C-488 decreased significantly when fibres were stretched (Fig. 4a).

Bottom Line: Heparin reduces binding but does not eliminate mechanosensitivity.The mechanical switch described here operates differently from the catch bond mechanism that Escherichia coli uses to adhere to surfaces under fluid flow.Demonstrating the existence of a mechanosensitive cell-binding site provides a new perspective on how the mechanobiology of ECM might regulate bacterial and cell-binding events, virulence and the course of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials, ETH Zurich, Zürich CH-8093, Switzerland.

ABSTRACT
Although soluble inhibitors are frequently used to block cell binding to the extracellular matrix (ECM), mechanical stretching of a protein fibre alone can physically destroy a cell-binding site. Here, we show using binding assays and steered molecular dynamics that mechanical tension along fibronectin (Fn) fibres causes a structural mismatch between Fn-binding proteins from Streptococcus dysgalactiae and Staphylococcus aureus. Both adhesins target a multimodular site on Fn that is switched to low affinity by stretching the intermodular distances on Fn. Heparin reduces binding but does not eliminate mechanosensitivity. These adhesins might thus preferentially bind to sites at which ECM fibres are cleaved, such as wounds or inflamed tissues. The mechanical switch described here operates differently from the catch bond mechanism that Escherichia coli uses to adhere to surfaces under fluid flow. Demonstrating the existence of a mechanosensitive cell-binding site provides a new perspective on how the mechanobiology of ECM might regulate bacterial and cell-binding events, virulence and the course of infection.

Show MeSH
Related in: MedlinePlus