Abstract
Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.
Highlights
Bacteria have developed a huge variety of sophisticated molecular strategies in order to colonize their hosts, to build up persistent infections, and to bypass the host’s defense mechanisms
Atomic force microscopy (AFM) cantilever tips functionalized with the FN constructs were repeatedly brought into contact with surfaces coated with CsgA to allow molecular bond formation (Fig. 1A–C)
We extended our single molecular force spectroscopy (SMFS) studies to directly measure the interaction forces of the FN constructs with fibrous curli expressed on intact living bacterial surfaces and placed Escherichia coli (E. coli) onto gelatin-coated mica surfaces[23]
Summary
Available AFM cantilevers (MSCT, Bruker, CA, USA) with a nominal spring constant of 0.01–0.03 N/m were functionalized with amino groups by using a 3-aminopropyltriethoxysilane (APTES) coating procedure[37]. The cantilevers were incubated for 5 min in 50 μl of Hepes-buffer saline (HBS) containing 2 μl of 5 mM NiCl2 to obtain a final concentration of 200 μM NiCl2. Bacterial suspensions were centrifuged (1500 ×g) for 2 min and the pellet was re-suspended in PBS buffer This washing step was repeated two times. Available tipless cantilevers (MLCT, Bruker, CA, USA) with a nominal spring constant of 0.03–0.1 N/m were functionalized with poly-L-lysine and glutaraldehyde to immobilize bacteria onto the cantilever. Force distance measurements were performed at room temperature (~25 °C) using tips of 0.01–0.03 N/m nominal spring constants, conjugated with fibronectin constructs (FN, FN III, RGD). Calculated de-adhesion works from individually measured force-distance curves were plotted in histograms and fitted with multi-Gaussian functions as shown in Figs 3E and 4D. 1000 force-distance curves from at least 5 different samples were taken to obtain the de-adhesion work distributions
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