Jorien Claes

Adhesion of Staphylococcus aureus to the vessel wall under flow is mediated by von Willebrand factor-binding protein

Adhesion of Staphylococcus aureus to blood vessels under shear stress requires von Willebrand factor (VWF). Several bacterial factors have been proposed to interact with VWF, including VWF-binding protein (vWbp), a secreted coagulase that activates the hosts prothrombin to generate fibrin. We measured the adhesion of S aureus Newman and a vWbp-deficient mutant (vwb) to VWF, collagen, and activated endothelial cells in a microparallel flow chamber. In vivo adhesion of S aureus was evaluated in the mesenteric circulation of wild-type (WT) and VWF-deficient mice. We found a shear-dependent increase in adhesion of S aureus to the (sub)endothelium that was dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was further enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets into bacterial microthrombi. In vivo, deficiency of vWbp or VWF as well as inhibition of coagulase activity reduced S aureus adhesion. We conclude that vWbp contributes to vascular adhesion of S aureus through 2 independent mechanisms: shear-mediated binding to VWF and activation of prothrombin to form S aureus-fibrin-platelet aggregates.

Plasminogen activation by staphylokinase enhances local spreading of S. aureus in skin infections

BackgroundStaphylococcus aureus (S. aureus) is a frequent cause of skin and soft tissue infections. A unique feature of S. aureus is the combined presence of coagulases that trigger fibrin formation and of the plasminogen activator staphylokinase (SAK). Whereas the importance of fibrin generation for S. aureus virulence has been established, the role of SAK remains unclear.We studied the role of plasminogen activation by SAK in a skin infection model in mice and evaluated the impact of alpha-2-antiplasmin (α2AP) deficiency on the spreading and proteolytic activity of S. aureus skin infections. The species-selectivity of SAK was overcome by adenoviral expression of human plasminogen. Bacterial spread and density was assessed non-invasively by imaging the bioluminescence of S. aureus Xen36.ResultsSAK-mediated plasmin activity increased the local invasiveness of S. aureus, leading to larger lesions with skin disruption as well as decreased bacterial clearance by the host. Even though fibrin and bacterial surfaces protected SAK-mediated plasmin activity from inhibition by α2AP, the deficiency of α2AP resulted in increased bacterial spreading. SAK-mediated plasmin also induced secondary activation of gelatinases, shown both in vitro and in lesions from the in vivo model.ConclusionSAK contributes to the phenotype of S. aureus skin infections by enhancing bacterial spreading as a result of fibrinolytic and proteolytic activation.

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis

BACKGROUND Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow. METHODS S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis. RESULTS Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves. CONCLUSIONS S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.

Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.

Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF‐binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A‐dependent surface protein Clumping factor A (ClfA). VWF‐vWbp‐ClfA anchor S. aureus to vascular endothelium under shear stress.

Bacterial adherence to graft tissues in static and flow conditions

Background Various conduits and stent‐mounted valves are used as pulmonary valve graft tissues for right ventricular outflow tract reconstruction with good hemodynamic results. Valve replacement carries an increased risk of infective endocarditis (IE). Recent observations have increased awareness of the risk of IE after transcatheter implantation of a stent‐mounted bovine jugular vein valve. This study focused on the susceptibility of graft tissue surfaces to bacterial adherence as a potential risk factor for subsequent IE. Methods Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguinis to bovine pericardium (BP) patch, bovine jugular vein (BJV), and cryopreserved homograft (CH) tissues was quantified under static and shear stress conditions. Microscopic analysis and histology were performed to evaluate bacterial adhesion to matrix components. Results In general, similar bacteria numbers were recovered from CH and BJV tissue surfaces for all strains, especially in flow conditions. Static bacterial adhesion to the CH wall was lower for S sanguinis adhesion (P < .05 vs BP patch). Adhesion to the BJV wall, CH wall, and leaflet was decreased for S epidermidis in static conditions (P < .05 vs BP patch). Bacterial adhesion under shear stress indicated similar bacterial adhesion to all tissues, except for lower adhesion to the BJV wall after S sanguinis incubation. Microscopic analysis showed the importance of matrix component exposure for bacterial adherence to CH. Conclusions Our data provide evidence that the surface composition of BJV and CH tissues themselves, bacterial surface proteins, and shear forces per se are not the prime determinants of bacterial adherence.

Inhibition of major integrin αVβ3 reduces Staphylococcus aureus attachment to sheared human endothelial cells.

Essentials Staphylococcus aureus (S. aureus) binds and impairs function of vascular endothelial cells (EC). We investigated the molecular signals triggered by S. aureus adhesion to EC. Inhibition of the EC integrin αVβ3 reduces S. aureus binding and rescues EC function. αVβ3 blockade represents an attractive target to treat S. aureus bloodborne infections.

In vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood. To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

Marginal role of von Willebrand factor-binding protein and coagulase in the initiation of endocarditis in rats with catheter-induced aortic vegetations

ABSTRACT Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman Δcoa, ΔvWbp, ΔclfA or Δcoa/ΔvWbp/ΔclfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion Δcoa/ΔvWbp/ΔclfA (incidence: 36%; P > 0.05 versus ΔclfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.

Assessment of the Dual Role of Clumping Factor A in S. Aureus Adhesion to Endothelium in Absence and Presence of Plasma

Adhesion of Staphylococcus aureus to endothelial cells (ECs) is paramount in infective endocarditis. Bacterial proteins such as clumping factor A (ClfA) and fibronectin binding protein A (FnbpA) mediate adhesion to EC surface molecules and (sub)endothelial matrix proteins including fibrinogen (Fg), fibrin, fibronectin (Fn) and von Willebrand factor (vWF). We studied the influence of shear flow and plasma on the binding of ClfA and FnbpA (including its sub-domains A, A16+, ABC, CD) to coverslip-coated vWF, Fg/fibrin, Fn or confluent ECs, making use of Lactococcus lactis, expressing these adhesins heterologously. Global adherence profiles were similar in static and flow conditions. In the absence of plasma, L. lactis-clfA binding to Fg increased with shear forces, whereas binding to fibrin did not. The degree of adhesion of L. lactis-fnbpA to EC-bound Fn and of L. lactis-clfA to EC-bound Fg, furthermore, was similar to that of L. lactis-clfA to coated vWF domain A1, in the presence of vWF-binding protein (vWbp). Yet, in plasma, L. lactis-clfA adherence to activated EC-vWF/vWbp dropped over 10 minutes by 80% due to vWF-hydrolysis by a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 and that of L. lactis-fnbpA likewise by > 70% compared to the adhesion in absence of plasma. In contrast, plasma Fg supported high L. lactis-clfA binding to resting and activated ECs. Or, in plasma S. aureus adhesion to active endothelium occurs mainly via two complementary pathways: a rapid but short-lived vWF/vWbp pathway and a stable integrin-coupled Fg-pathway. Hence, the pharmacological inhibition of ClfA-Fg interactions may constitute a valuable additive treatment in infective endocarditis.