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

BACKGROUND: Vascular endothelial dysfunction with associated oedema and organ failure is one of the hallmarks of sepsis. While a large number of microorganisms can cause sepsis, Staphylococcus aureus is one of the primary etiological agents. Currently there are no approved specific treatments for sepsis and therefore the initial management bundle focuses on cardiorespiratory resuscitation and mitigation against the immediate threat of uncontrolled infection. The continuous emergence of antibiotic resistant strains of bacteria urges the development of new therapeutic approaches for this disease. OBJECTIVE: The objective of this study was to identify the molecular mechanisms leading to endothelial dysfunction as a result of Staphylococcus aureus binding. METHODS: Stahpylococcus aureus Newman and clumping factor A-deficient binding to endothelium were measured in vitro and in the mesenteric circulation of C57Bl/6 mice. The effect of the αVβ3 blocker, cilengitide, on bacterial binding, endothelial VE-cadherin expression, apoptosis, proliferation and permeability were assessed. RESULTS: Here we show that the major Staphylococcus aureus cell wall protein clumping factor A binds to endothelial cell integrin αVβ3 in the presence of fibrinogen. This interaction results in disturbances in barrier function mediated by VE-cadherin in endothelial cell monolayers and ultimately cell death by apoptosis. Using a low concentration of cilengitide, ClfA binding to αVβ3 was significantly inhibited both in vitro and in vivo. Moreover, preventing Staphylococcus aureus from attaching to αVβ3 resulted in a significant reduction in endothelial dysfunction following infection. CONCLUSION: Inhibition of Staphylococcus aureus ClfA binding to endothelial cell αVβ3 using cilengitide prevents endothelial dysfunction. This article is protected by copyright. All rights reserved

The impact of flow on Staphylococcus aureus - endothelium interactions in infective endocarditis

Infective endocarditis is an infection of the inner surface of the heart, most frequently of the heart valves. Mortality due to infective endocarditis remains high despite adequate and timely treatment. Staphylococcus aureus (S. aureus) is one of the leading causes of infective endocarditis and compared to other pathogens infective endocarditis by S. aureus results in a higher mortality. It is more frequently associated with severe and lethal complications. The increasing number of antibiotic-resistant S. aureus strains urges for new therapeutic approaches to reduce S. aureus infections and disease severity. Given the aggressive nature of S. aureus infective endocarditis strategies targeting key virulence factors involved in the early phase of the infectious process are an attractive option to achieve this goal. To establish endovascular infections and infective endocarditis in particular, spreading of S. aureus requires a mechanism that allows its adhesion to the blood vessel wall and that can overcome the shear stress exerted by rapidly flowing blood. A potential role for von Willebrand factor (VWF) in S. aureus adhesion to blood vessels under flow has recently been recognized. Several bacterial factors have been proposed to interact with VWF, including von Willebrand factor-binding protein (vWbp), a secreted coagulase that contributes to S. aureus pathophysiology by activating the host’s prothrombin. However, the mechanism through which S. aureus interacts with VWF under shear forces remained elusive. S. aureus expresses a number of bacterial cell wall-anchored proteins that mediate bacterial adhesion to the host and contribute to the pathogenesis of endovascular infections, e.g. the well studied fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA). Most of these proteins are positioned in the bacterial cell wall by a mechanism that involves cleavage of a conserved Leu-Pro-X-Thr-Gly (LPXTG) motif. A mutation in the srtA gene (sortase A) leads to an anchoring defect in about 20 S. aureus cell wall-anchored proteins. Yet, the underlying molecular pathways that control bacterial adherence, especially in a flow field of high shear stress, including the contribution of individual receptors to bacterial recruitment were still poorly understood at the start of this PhD work. Therefore, in the present study we focused on the adherence of S. aureus to endothelial cells and subendothelial matrix under flow conditions. We developed and adopted an in vitro perfusion model to study bacterial adhesion to coated surfaces under different shear rates and an in vivo mesenteric perfusion model to study real-time bacterial adhesion to the murine mesenteric circulation. Our studies revealed a shear-dependent increase of S. aureus adhesion to (sub)endothelium dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets via the receptor αΙΙbβ3 to form microthrombi. Correspondingly, coagulase inhibition and inhibition of platelet αΙΙbβ3 reduced the adhesion of S. aureus to (sub)endothelium. In vivo, deficiency of vWbp or VWF and inhibition of coagulase activity reduced S. aureus adhesion to the vessel wall. vWbp is thought to be a secreted protein not anchoring to the bacterial cell wall. Therefore, we further investigated how vWbp mediates bacterial adhesion and we identified ClfA, a SrtA-mediated surface protein, as the bacterial surface binding partner for vWbp. Absence of SrtA or ClfA reduced adhesion of S. aureus to vWbp, VWF A1-domain and activated endothelial cells. The selective overexpression of ClfA in the membrane of Lactococcus lactis (L. lactis) enabled these bacteria to bind to vWbp and to the VWF A1-domain but only in the presence of exogenously added staphylococcal vWbp. Absence of ClfA in the S. aureus ClfA mutant abolished bacterial adhesion to the activated mesenteric vessel wall of C57Bl6 mice. Comparing static in vitro and in vivo experiments reveals a different role of bacterial binding via fibronectin or fibrinogen suggesting an impact of shear stress on major bacterial binding properties. We investigated the contributing role of ClfA, FnBPA and FnBPA domains in bacterial adhesion under flow to fibronectin, fibrinogen and endothelial cells with emphasis on interactions with fibrin, always massively present in endocarditis lesions. The adhesion to fibrin was primarily, but not exclusively assured by ClfA, whereas both FnBPA and ClfA contributed to L. lactis adhesion to endothelium in an Fn and Fg dependent manner, respectively.status: publishe

Endothelial cells response to staphylococcus aureus: critical role of clumping Factor A in sepsis

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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.status: publishe

Binding to Von Willebrand Factor enable Staphylocccus aureus to overcome shear stress and cause infective endocarditis

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Inhibition of major integrin alpha(V)beta(3) 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. SUMMARY: Background Vascular endothelial dysfunction with associated edema and organ failure is one of the hallmarks of sepsis. Although a large number of microorganisms can cause sepsis, Staphylococcus aureus (S. aureus) is one of the primary etiologic agents. Currently, there are no approved specific treatments for sepsis, and the initial management bundle is therefore focused on cardiorespiratory resuscitation and mitigation of the immediate threat of uncontrolled infection. The continuous emergence of antibiotic-resistant strains of bacteria necessitates the development of new therapeutic approaches for this disease. Objective To identify the molecular mechanisms leading to endothelial dysfunction as a result of S. aureus binding. METHODS: Binding of wild type and Clumping factor A (ClfA) deficient S. aureus Newman to the endothelium was measured in vitro and in the mesenteric circulation of C57Bl/6 mice. The effects of the αV β3 blocker-cilengitide-on bacterial binding, endothelial VE-cadherin expression, apoptosis, proliferation and permeability were assessed. Results The major S. aureus cell wall protein ClfA bound to endothelial cell αV β3 in the presence of fibrinogen. This interaction resulted in disturbances in barrier function mediated by VE-cadherin in endothelial cell monolayers, and ultimately cell death by apoptosis. With a low concentration of cilengitide, ClfA binding to αV β3 was significantly inhibited both in vitro and in vivo. Moreover, preventing S. aureus from attaching to αV β3 resulted in a significant reduction in endothelial dysfunction following infection. Conclusion Inhibition of S. aureus ClfA binding to endothelial cell αV β3 by cilengitide prevents endothelial dysfunction.status: publishe

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 host's 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.status: publishe

Clumping factor A is a membrane receptor for secreted von Willebrand factor-binding protein mediating Staphylococcus aureus adhesion to vascular endothelium

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