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 binding of bacteria to human platelets is thought to be a central event in the development of endocarditis (a life-threatening cardiovascular infection). We have previously found that platelet binding by Streptococcus mitis is mediated by surface components encoded by a bacteriophage contained within the host bacterium. We now show that lysin (an enzyme of bacteriophage origin) contributes to platelet binding via its direct interaction with fibrinogen on the platelet surface. Lysin bound to purified fibrinogen in vitro, and this interaction specifically involved the Aα and Bβ chains of fibrinogen. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the gene encoding lysin gene resulted in a significant reduction in binding to fibrinogen by S. mitis, as well as a major reduction in virulence, as measured by a rat model of endocarditis. These results indicate that lysin is a multifunctional protein, representing a new class of fibrinogen-binding molecules. Lysin is localized to the bacterial surface via its interaction with cell wall choline, where it then can bind fibrinogen directly. Cell surface lysin apparently also contributes to the development of endovascular infections via its previously unrecognized fibrinogen binding activity.