Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 05 Aug 2019 11:43:47 Direct interaction of iron-regulated surface determinant IsdB of Staphylococcus aureus with the GPIIb/IIIa receptor on platelets Helen Miajlovic, 1 Marta Zapotoczna, 1 Joan A. Geoghegan, 1 Steven W. Kerrigan, 2 Pietro Speziale 3 and Timothy J. Foster 1 Correspondence Timothy J. Foster tfoster@tcd.ie 1 Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland 2 Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2, Ireland 3 Department of Biochemistry, Viale Taramelli 3/b, 27100 Pavia, Italy Received 24 November 2009 Accepted 9 December 2009 The interaction of bacteria with platelets is implicated in the pathogenesis of endovascular infections, including infective endocarditis, of which Staphylococcus aureus is the leading cause. Several S. aureus surface proteins mediate aggregation of platelets by fibrinogen- or fibronectin- dependent processes, which also requires specific antibodies. In this study S. aureus was grown in iron-limited medium to mimic in vivo conditions in which iron is unavailable to pathogens. Under such conditions, a S. aureus mutant lacking the known platelet-activating surface proteins adhered directly to platelets in the absence of plasma proteins and triggered aggregation. Platelet adhesion and aggregation was prevented by inhibiting expression of iron-regulated surface determinant (Isd) proteins. Mutants defective in IsdB, but not IsdA or IsdH, were unable to adhere to or aggregate platelets. Antibodies to the platelet integrin GPIIb/IIIa inhibited platelet adhesion by IsdB-expressing strains, as did antagonists of GPIIb/IIIa. Surface plasmon resonance demonstrated that recombinant IsdB interacts directly with GPIIb/IIIa. INTRODUCTION Staphylococcus aureus is an opportunistic pathogen that colonizes the desquamated epithelium of the anterior nares. At sites of infection such as wounds, ulcers or abscesses, S. aureus can gain access to the bloodstream, resulting in bacteraemia. Increased use of intravascular devices and invasive procedures in hospitals has led to an increased incidence of bacteraemic infections (Moreillon & Que, 2004). Several endovascular infections, including infective endocarditis, result from the interaction of bacteria with platelets. S. aureus is the leading cause of infective endocarditis, which is characterized by the build- up, on heart-valve surfaces, of vegetative bodies consisting of bacteria, fibrin and aggregated platelets (Moreillon & Que, 2004; Mylonakis & Calderwood, 2001). The ability of S. aureus and other bacteria to adhere to and aggregate platelets is thought to contribute to the development of endovascular infections. Aggregation of platelets by bacteria is the result of a multi-step process. Bacteria interact with receptors on the platelet surface either directly or indirectly through bridging molecules such as fibrinogen. Initial adhesion of bacteria to platelets can result in subsequent platelet activation, which is characterized by signalling events and calcium oscillations within the platelet. Upon activation, the major platelet integrin GPIIb/IIIa undergoes conformational changes that allow it to bind avidly to fibrinogen and fibronectin in solution (Calvete, 1999). Aggregation of platelets occurs when adjacent platelets interact with the c-chain of the bivalent fibrinogen molecule, cross-linking platelets into aggregates. The interaction between S. aureus and platelets is complex and involves multiple factors (O’Brien et al., 2002). Several surface-expressed proteins can stimulate platelet aggrega- tion. These include the fibrinogen-binding proteins clumping factors A and B (ClfA and ClfB), and the bifunctional fibronectin-fibrinogen binding proteins FnBPA and FnBPB (Fitzgerald et al., 2006b; Loughman et al., 2005; Miajlovic et al., 2007). Recent studies have shown that, under high shear rates such as those seen in small arteries and arterioles, protein A and ClfA are crucial for platelet aggregation (Kerrigan et al., 2008; Pawar et al., 2004). Binding of fibrinogen or fibronectin by surface proteins effectively coats S. aureus with these two ligands, allowing it to engage the low-affinity form of the GPIIb/IIIa on resting platelets. Activation of platelets requires specific antibodies Abbreviations: GFP, gel-filtered platelets; PRP, platelet-rich plasma; WP, washed platelets. Microbiology (2010), 156, 920–928 DOI 10.1099/mic.0.036673-0 920 036673 G 2010 SGM Printed in Great Britain