First analysis of a bacterial collagen-binding protein with collagen Toolkits: promiscuous binding of YadA to collagens may explain how YadA interferes with host processes

Abstract

The Yersinia adhesin YadA mediates the adhesion of the human enteropathogen Yersinia enterocolitica to collagens and other components of the extracellular matrix. Though YadA has been proposed to bind to a specific site in collagens, the exact binding determinants for YadA in native collagen have not previously been elucidated. We investigated the binding of YadA to collagen Toolkits, which are libraries of triple-helical peptides spanning the sequences of type II and III human collagens. YadA bound to many of them, in particular to peptides rich in hydroxyproline but with few charged residues. We were able to block the binding of YadA to collagen type IV with the triple-helical peptide (Pro-Hyp-Gly)(10), suggesting that the same site in YadA binds to triple-helical regions in network-forming collagens as well. We showed that a single Gly-Pro-Hyp triplet in a triple-helical peptide was sufficient to support YadA binding, but more than six triplets were required to form a tight YadA binding site. This is significantly longer than the case for eukaryotic collagen-binding proteins. YadA-expressing bacteria bound promiscuously to Toolkit peptides. Promiscuous binding could be advantageous for pathogenicity in Y. enterocolitica and, indeed, for other pathogenic bacteria. Many of the tightly binding peptides are also targets for eukaryotic collagen-binding proteins, and YadA was able to inhibit the interaction between selected Toolkit peptides and platelets. This leads to the intriguing possibility that YadA may interfere in vivo with host processes mediated by endogenous collagen-binding proteins.

FIG. 1.

Binding of YadA to collagen Toolkits in SPBA. Wells of microtiter plates were coated with Toolkit peptides and probed with YadA. Panel A shows the binding of YadA to Toolkit II, and panel B shows the binding of YadA to Toolkit III. Collagens of the appropriate type, GPP10, and GFOGER were included as controls. A triple-helical conformation is known to be necessary for YadA binding (30). Binding to BSA shows background levels. Toolkit III contains an internal control for the triple-helical conformation, as peptides III-6 and III-52 do not form a collagenous triple helix, unlike the other Toolkit peptides (28, 43). As expected, YadA bound to neither of these peptides. The columns show the mean absorbances at 450 nm for three replicate wells; error bars denote standard errors of the means.

FIG. 2.

Distributions of Toolkit peptides sorted according to YadA binding response. Toolkit peptides were classed as nonbinders (response of <0.15 AU at 450 nm; white columns), low binders (response between 0.15 and 0.4 AU; striped columns), intermediate binders (response of >0.4, but below that of the corresponding collagen; gray columns), and high binders (response above that of the corresponding collagen; black columns). The peptides were sorted according to mean absorbances at 450 nm for three replicate wells, as shown in Fig. 1. Panel A shows the sorted peptides from Toolkit II and panel B the sorted peptides from Toolkit III.

FIG. 3.

Binding of YadA-expressing bacteria to Toolkit peptides in SPBA. Bacteria were incubated in wells of a microtiter plate coated with a subset of Toolkit peptides. The results show the mean absorbances at 493 nm for three replicate wells; error bars denote standard errors of the means. YeO3 is a YadA-positive strain, whereas YeO3-c does not express YadA. Collagen type I, GPP10, and BSA were included as controls.

FIG. 4.

Binding of YadA to GPO peptides in SPBA. Wells of a microtiter plate were coated with peptides with different numbers of GPO repeats (GPO1 to GPO6) or with different spacings between the same number of GPO triplets (GPO6a to -j) and then were probed with YadA. As controls, we included (POG)₁₀, (POG)₅, GPP10, and the non-triple-helical peptide Gly⁻, which is the same as (POG)₁₀ but lacks a central glycine residue (32). Collagen type I was included to assess binding levels. The BSA column shows background levels. The columns show the mean absorbances at 490 nm for three replicate wells; error bars denote standard errors of the means. The difference between GPP10 and GPO1 shows that a single GPO triplet in a GPP background is sufficient for tight binding.

FIG. 5.

Binding of YadA to GKO-GPO peptides in SPBA. Wells of a microtiter plate were coated with peptides with different numbers of GPO repeats flanked on either side by GKO and then were probed with YadA. The peptides GPO1, GPO4, GPO6, GPP10, and Gly⁻ were used as controls. The BSA column shows background levels. The columns show the mean absorbances at 450 nm for three replicate wells; error bars denote standard errors of the means.

FIG. 6.

Inhibition of platelet adhesion to collagen type I or collagen-like peptides by YadA. Coverslips were coated with collagen type I or a mixture or three collagen-like peptides. Blood (incubated with DIOC6 to stain platelets) was drawn across coated coverslips at an arterial shear rate (1,000 s⁻¹), and the adhesion of platelets was determined by confocal microscopy. (A) Surface coverage (%) by platelets. (B) Mean height of thrombi. (C) ZV₅₀ value, i.e., the mean height of the center of mass of the thrombi, a value that measures platelet activation. Results from control experiments without YadA (−YadA) are shown with black columns. White columns show the results from experiments where the coverslips were previously incubated with YadA (+YadA). The columns represent the mean values for four replicates, with standard errors of the means. (D) Representative confocal microscopy images of platelets binding to coverslips coated with collagen type I or a mixture of three collagen-like peptides. The lower row shows the effects of blocking with YadA (100 μg/ml) before running blood over the coverslips; the upper row shows controls.