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