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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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Experimental validation of mechanosensitive binding of bacterial peptides to Fn fibres.(a, b) Strain-dependent binding of bacterial peptides B3C-Alexa488 and STAFF5C-Alexa488 to Cy5-labelled Fn fibres. The mean of the intensity ratios (IB3C-488/IFn-Cy5) of relaxed fibres was set to 1 and the other values were scaled accordingly. All mean values (shown by black bar) are significantly different from each other with P<0.001 (unpaired two-tailed Student's t-test), except where noted. (a) Intensity ratio plot of Alexa488-labelled B3C to Cy5-labelled Fn fibres versus Fn fibre strain (see Fig. 3). Each experiment (shown as differently shaped and coloured data points) consisted of 30 fibres: 10 were relaxed (∼7% strain), 10 deposited only in a prestrained state (no mechanical manipulation of the silicone sheet, ∼140% strain26) and 10 stretched (∼300 or ∼380% strain). (b) Intensity ratio plot of Alexa488-labelled STAFF5C to Cy5-labelled Fn fibres versus Fn fibre strain. Each experiment (shown as differently shaped and coloured data points) consisted of 40 fibres, including 20 relaxed (∼0 and ∼100% strain) and 20 stretched fibres (∼250 and ∼380% strain). Inhibition of strain-dependent binding of the bacterial peptide B3C-Alexa488 to Cy5-labelled Fn fibres in the presence (red bars) of (c) soluble Fn (300 μl mg−1) and (d) heparin (100 μl mg−1). The mean of the intensity ratio of relaxed fibres in the absence (blue bars) of soluble Fn or heparin was set to 1 and the remaining three values were scaled accordingly. All values are significantly different from each other (unpaired two-tailed Student's t-test) with P<0.0001, except where indicated. Values are means of intensity ratios of 20 or 10 fibres in the presence of Fn or heparin, respectively, and error bars indicate s.d.
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f4: Experimental validation of mechanosensitive binding of bacterial peptides to Fn fibres.(a, b) Strain-dependent binding of bacterial peptides B3C-Alexa488 and STAFF5C-Alexa488 to Cy5-labelled Fn fibres. The mean of the intensity ratios (IB3C-488/IFn-Cy5) of relaxed fibres was set to 1 and the other values were scaled accordingly. All mean values (shown by black bar) are significantly different from each other with P<0.001 (unpaired two-tailed Student's t-test), except where noted. (a) Intensity ratio plot of Alexa488-labelled B3C to Cy5-labelled Fn fibres versus Fn fibre strain (see Fig. 3). Each experiment (shown as differently shaped and coloured data points) consisted of 30 fibres: 10 were relaxed (∼7% strain), 10 deposited only in a prestrained state (no mechanical manipulation of the silicone sheet, ∼140% strain26) and 10 stretched (∼300 or ∼380% strain). (b) Intensity ratio plot of Alexa488-labelled STAFF5C to Cy5-labelled Fn fibres versus Fn fibre strain. Each experiment (shown as differently shaped and coloured data points) consisted of 40 fibres, including 20 relaxed (∼0 and ∼100% strain) and 20 stretched fibres (∼250 and ∼380% strain). Inhibition of strain-dependent binding of the bacterial peptide B3C-Alexa488 to Cy5-labelled Fn fibres in the presence (red bars) of (c) soluble Fn (300 μl mg−1) and (d) heparin (100 μl mg−1). The mean of the intensity ratio of relaxed fibres in the absence (blue bars) of soluble Fn or heparin was set to 1 and the remaining three values were scaled accordingly. All values are significantly different from each other (unpaired two-tailed Student's t-test) with P<0.0001, except where indicated. Values are means of intensity ratios of 20 or 10 fibres in the presence of Fn or heparin, respectively, and error bars indicate s.d.

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)

Experimental validation of mechanosensitive binding of bacterial peptides to Fn fibres.(a, b) Strain-dependent binding of bacterial peptides B3C-Alexa488 and STAFF5C-Alexa488 to Cy5-labelled Fn fibres. The mean of the intensity ratios (IB3C-488/IFn-Cy5) of relaxed fibres was set to 1 and the other values were scaled accordingly. All mean values (shown by black bar) are significantly different from each other with P<0.001 (unpaired two-tailed Student's t-test), except where noted. (a) Intensity ratio plot of Alexa488-labelled B3C to Cy5-labelled Fn fibres versus Fn fibre strain (see Fig. 3). Each experiment (shown as differently shaped and coloured data points) consisted of 30 fibres: 10 were relaxed (∼7% strain), 10 deposited only in a prestrained state (no mechanical manipulation of the silicone sheet, ∼140% strain26) and 10 stretched (∼300 or ∼380% strain). (b) Intensity ratio plot of Alexa488-labelled STAFF5C to Cy5-labelled Fn fibres versus Fn fibre strain. Each experiment (shown as differently shaped and coloured data points) consisted of 40 fibres, including 20 relaxed (∼0 and ∼100% strain) and 20 stretched fibres (∼250 and ∼380% strain). Inhibition of strain-dependent binding of the bacterial peptide B3C-Alexa488 to Cy5-labelled Fn fibres in the presence (red bars) of (c) soluble Fn (300 μl mg−1) and (d) heparin (100 μl mg−1). The mean of the intensity ratio of relaxed fibres in the absence (blue bars) of soluble Fn or heparin was set to 1 and the remaining three values were scaled accordingly. All values are significantly different from each other (unpaired two-tailed Student's t-test) with P<0.0001, except where indicated. Values are means of intensity ratios of 20 or 10 fibres in the presence of Fn or heparin, respectively, and error bars indicate s.d.
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f4: Experimental validation of mechanosensitive binding of bacterial peptides to Fn fibres.(a, b) Strain-dependent binding of bacterial peptides B3C-Alexa488 and STAFF5C-Alexa488 to Cy5-labelled Fn fibres. The mean of the intensity ratios (IB3C-488/IFn-Cy5) of relaxed fibres was set to 1 and the other values were scaled accordingly. All mean values (shown by black bar) are significantly different from each other with P<0.001 (unpaired two-tailed Student's t-test), except where noted. (a) Intensity ratio plot of Alexa488-labelled B3C to Cy5-labelled Fn fibres versus Fn fibre strain (see Fig. 3). Each experiment (shown as differently shaped and coloured data points) consisted of 30 fibres: 10 were relaxed (∼7% strain), 10 deposited only in a prestrained state (no mechanical manipulation of the silicone sheet, ∼140% strain26) and 10 stretched (∼300 or ∼380% strain). (b) Intensity ratio plot of Alexa488-labelled STAFF5C to Cy5-labelled Fn fibres versus Fn fibre strain. Each experiment (shown as differently shaped and coloured data points) consisted of 40 fibres, including 20 relaxed (∼0 and ∼100% strain) and 20 stretched fibres (∼250 and ∼380% strain). Inhibition of strain-dependent binding of the bacterial peptide B3C-Alexa488 to Cy5-labelled Fn fibres in the presence (red bars) of (c) soluble Fn (300 μl mg−1) and (d) heparin (100 μl mg−1). The mean of the intensity ratio of relaxed fibres in the absence (blue bars) of soluble Fn or heparin was set to 1 and the remaining three values were scaled accordingly. All values are significantly different from each other (unpaired two-tailed Student's t-test) with P<0.0001, except where indicated. Values are means of intensity ratios of 20 or 10 fibres in the presence of Fn or heparin, respectively, and error bars indicate s.d.
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