Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

Abstract

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis.

The human gut virome: Inter-individual variation and dynamic response to diet

“…Also seen were the expected diversity of lysins (Seo et al 2010), holins (Wang et al 2000), bacteriocins (Dawid et al 2007), restriction/modification systems (Kobayashi 2001), and virulence factors (O'Brien et al 1984;Lainhart et al 2009;Campos et al 2010).…”

Platelet-Bacterial Interactions in the Pathogenesis of Infective Endocarditis — Part I: The Streptococcus

“…It is likely that the initial adhesion events occur independently of platelet activation and thus make suitable targets in the prevention of IE. In contrast, as the ability to induce platelet aggregation (activation) in vitro contributes to the virulence and persistence of the organism in infective endocarditis animal models [87,95], the pathways of platelet aggregation are targets of future IE therapies. Consistent with this, a recent study has examined the effect of antiplatelet drug Reopro (abciximab) in the treatment of sepsis in mice [113] and the use of cyclooxygenase inhibitors, e.g.…”

“…When investigated further, the phage lysin was found to bind fibrinogen via the D fragment of the Aα and Bβ chains, and in doing so can mediate an indirect interaction with human platelets through αIIbβ3 [87]. Like PblA and PblB, it is also a choline binding protein but with homology to the choline binding domain of pneumococcal LytA [87]. The fibrinogen interactive domain was localized to amino acids 102-198 [88] and when this polypeptide was preincubated with platelets and S. mitis SF100, it significantly extended the lag time to aggregation.…”

“…Furthermore, in a rat model of endocarditis, co-infection with PblA . Lysin SM1 is considered a multifunctional phage protein, mediating lysis of S. mitis in the bacteriophage lytic life cycle, binding to choline residues in the cell wall and binding to fibrinogen, bridging an interaction with human platelets via αIIbβ3 [87], an interaction that is repeated in multiple streptococcal species and considered an important interaction in the pathogenesis of IE (see figure).…”

Platelet-Bacterial Interactions as Therapeutic Targets in Infective Endocarditis

“…S. mitis strains lacking the expression of PblA and PblB demonstrated significant decrease in platelet binding invitro as well as marked reduction in virulence in an animal model of infective endocarditis (Bensing et al, 2001b;Mitchell et al, 2007). More recent studies have demonstrated that S. mitis surface bound lysin can bind both free and platelet bound fibrinogen, through its interaction with the Aa and Bb chains of fibrinogen (Seo et al, 2010). Once S. mitis has bound fibrinogen this in turn can bind to the platelet fibrinogen receptor, GPIIbIIIa, initiating thrombus formation.…”