Streptococcus Gordonii Binds VWF Via Its Sialoglycan-Binding Adhesins Under Shear to Initiate Infective Endocarditis

Introduction: Streptococcus gordonii (SG), a commensal bacterium of the oral cavity, is a significant cause of native-valve (without prior valvular defect) and subacute infective endocarditis (IE), often attributed to its extraordinary ability to form biofilms. IE pathogenesis requires bacterial adhesion to the valve, which may involve binding to platelet (PLT) glycoprotein (GP) Ibα through SG serine-rich repeat (SRR) adhesins. In addition, the SG receptor PadA binds platelet integrin α_IIbβ₃. Binding to von Willebrand factor (VWF) has been shown for staphylococci but not for SG. The SG strain expressing the SRR adhesin GspB binds core 1 (C1) sialoglycans on the mechanosensory domain of GPIbα, 10712BR adhesin binds core 2 (C2) sialoglycans on the mucin stalk. Hsa adhesin binds both C1 and C2. Pathogenicity is at least partially determined by glycan-binding specificity. In a rat model, selective C1 binding results in enhanced virulence, contributing to infection initiation and progression. Here, we investigate the impact of glycan ligand specificity and the role of shear in SG binding to GPIbα and VWF, which also has C1 sialoglycans.

Methods: Whole blood (WB), platelet-rich plasma (PRP), or washed PLTs were obtained from healthy donors (n = 29, 55% females). SG-PLT binding and PLT activation following a static 10 min incubation were measured by flow cytometry and under shear conditions using microfluidics, in isogenic SG strains (C1, C2, C1&2, and no (C0)-sialoglycan binding).

Results:

SG binding to blood cells, static. In WB, the C2 binder showed the highest binding to PLT (~70%), followed by C1&2 (~40%), with C1 and C0 showing minimal binding. WBC binding showed a different pattern; RBC binding was minimal. Between 15 and 40% of each strain bound blood cells with C2 and C1&2 binders showing a higher bound-fraction due to PLT binding. Previously reported plasma protein ligands of SG adhesins may account for the remainder of the binding. The same pattern of binding was seen in PRP as in WB. However, in washed PLTs, binding was ~3x higher, indicating that plasma contains inhibitory factors. C2 and C1&2-binders bound similarly. Glycocalicin or WM23 (mAb to the mucin stalk) can partially inhibit this binding, while SZ2 (mAb to GPIbα sulfo-tyrosine region) promotes binding.

Effect of SG on platelet activation, static. In washed PLTs, C2 and C1&2 binders induced higher P-selectin expression (p<0.0001 and p=0.0001, respectively) and phosphatidylserine exposure compared to baseline (p = 0.03). In PRP, there were no apparent differences in PLT activation between strains. In PRP lumi-aggregometry, SG did not cause spontaneous PLT aggregation or ATP secretion. However, in the presence of minimal concentrations of ADP (1 µM), arachidonic acid (0.1 mM), or collagen (0.2 μg/mL), SG strains similarly induced maximal PLT aggregation.

Studies under shear. We evaluated by microfluidics whether the binding of SG to PLT or VWF is shear-dependent. We first monitored adhesion of SG and PLT in WB perfused into VWF-coated channels. The C1-selective binder adhered more extensively to immobilized VWF than the other strains, independently of PLT adhesion, at both 500 and 2,000 s^-1 shear rates. At 2,000 s^-1, addition of SG induced greater PLT adhesion than without SG. We also studied the effect of SG on platelet thrombus formation in WB by perfusing the mixture into collagen-coated block-and-post channels, which provides high turbulent shear (24,000 s^-1). C1 and C1&2 binding strains induced much larger thrombi, with greater SG incorporation. To evaluate SG-VWF binding, we perfused the bacteria over self-associated purified VWF (mono-pillar channels) and endothelium-secreted VWF (flow chambers with cultured HUVECs). We observed higher binding of the C1-binder to VWF strings around the pillar, possibly enhancing VWF self-association. On HUVECs, all SG strains except C0 formed beads-on-a-string structures on VWF, without colocalization with PLTs.

Conclusion: The study shows a previously undescribed shear-dependent interaction between SG and VWF via SG’s sialoglycan-binding SRR adhesin with C1 specificity. This provides a likely explanation for how the bacteria attach to heart valves at high shear rates to initiate infection and also suggests a reason for the increased virulence of the C1-binding strains. The C2-binding strains bind platelets through GPIbα, which may enhance bacterial clearance and reduce virulence.