Inhibition of Fibrinolysis by Streptococcal Phage LysinSM1

Abstract

Expression of bacteriophage lysin_SM1 by Streptococcus oralis strain SF100 is thought to be important for the pathogenesis of infective endocarditis, due to its ability to mediate bacterial binding to fibrinogen. To better define the lysin_SM1 binding site on fibrinogen Aα, and to investigate the impact of binding on fibrinolysis, we examined the interaction of lysin_SM1 with a series of recombinant fibrinogen Aα variants. These studies revealed that lysin_SM1 binds the C-terminal region of fibrinogen Aα spanned by amino acid residues 534 to 610, with an affinity of equilibrium dissociation constant (K_D) of 3.23 × 10^-5 M. This binding site overlaps the known binding site for plasminogen, an inactive precursor of plasmin, which is a key protease responsible for degrading fibrin polymers. When tested in vitro, lysin_SM1 competitively inhibited plasminogen binding to the αC region of fibrinogen Aα. It also inhibited plasminogen-mediated fibrinolysis, as measured by thromboelastography (TEG). These results indicate that lysin_SM1 is a bi-functional virulence factor for streptococci, serving as both an adhesin and a plasminogen inhibitor. Thus, lysin_SM1 may facilitate the attachment of bacteria to fibrinogen on the surface of damaged cardiac valves and may also inhibit plasminogen-mediated lysis of infected thrombi (vegetations) on valve surfaces. IMPORTANCE The interaction of streptococci with human fibrinogen and platelets on damaged endocardium is a central event in the pathogenesis of infective endocarditis. Streptococcus oralis can bind platelets via the interaction of bacteriophage lysin_SM1 with fibrinogen on the platelet surface, and this process has been associated with increased virulence in an animal model of endocarditis. We now report that lysin_SM1 binds to the αC region of the human fibrinogen Aα chain. This interaction blocks plasminogen binding to fibrinogen and inhibits fibrinolysis. In vivo, this inhibition could prevent the lysis of infected vegetations, thereby promoting bacterial persistence and virulence.

Keywords: Streptococcus mitis; fibrinogen; fibrinolysis; infective endocarditis; plasminogen; thromboelastography.

FIG 1

Schematic diagram of the intact fibrinogen dimer. The individual chains, Aα, Bβ, and γ, are blue, green, and red, respectively. Fibrinopeptides A and B (FpA and FpB) are magenta, and the disulfide bonds are shown by black bars. αC domains are consistent with the αC-domain and the flexible αC-connectors.

FIG 2

Identification of lysin_SM1 and plasminogen binding regions on the fibrinogen Aα chain. (A) Schematic diagram of the fibrinogen Aα chain and its recombinant variants. All variants were expressed as MalE fusion proteins. (B) Binding of lysin_102-198 (10 μg) or plasminogen (10 μg) to human fibrinogen Aα and Aα variants 2 and 3; analysis by far Western blotting. (C) Binding of lysin_102-198 or plasminogen to immobilized recombinant fibrinogen Aα variants 3 to 8 (0.1 μM). Recombinant fibrinogen truncates were separated by SDS-PAGE and stained with Coomassie blue (top). * indicates the expected molecular sizes of recombinant variants. The binding region was identified by measuring lysin_102-198 (1 μM; middle) or plasminogen (1 μM; bottom) binding to immobilized fibrinogen truncates. Bound proteins were detected with anti-FLAG or anti-plasminogen monoclonal antibodies.

FIG 3

Quantitative analysis of fibrinogen binding by lysin_SM1, plasminogen, and streptococci. (A) Binding of _FLAGlysin_102-198 (left) and plasminogen (right) to immobilized fibrinogen Aα variants 4, 5, or 8 (0.1 μM), as measured by ELISA, using either anti-FLAG or anti-plasminogen antibody. (B) Inhibition of lysin_SM1 binding to immobilized human fibrinogen, recombinant fibrinogen Aα variant 8, or variant 5. * ,  P < 0.05 compared with the untreated group. (C) Relative binding of S. oralis SF100 WT or its isogenic Δlysin mutant (PS1006) to immobilized fibrinogen or fibrinogen Aα variants 8 or 5. Bacteria (10⁹ CFU/ml) were incubated with immobilized fibrinogen or fibrinogen Aα variant 8 or 5 (0.1 μM). Bound bacteria were stained with 0.1% crystal violet and measured for optical density at 595 nm. Bars indicate the means (±SD) of triplicate results from a representative experiment. *,  P < 0.05 compared with WT.

FIG 4

Interaction of plasminogen and lysin_102-198 with fibrinogen and the fibrinogen αC region. (A and B) Surface plasmon resonance analysis of lysin_SM1 (left panel), lysin_102-198 (middle panel), and plasminogen (right panel) binding to (A) fibrinogen and (B) variant 8.

FIG 5

Inhibition of plasminogen binding to fibrinogen by recombinant lysin_SM1 and secreted lysin_SM1. (A) Recombinant lysin_SM1 inhibits plasminogen binding to immobilized fibrinogen. Plasminogen was preincubated with recombinant lysin_SM1, lysin_102-198, or lysin_1-102 (0, 0.8, 4, 20, and 100 μM) and transferred to 96-well plates containing immobilized fibrinogen (0.1 μM). Bound plasminogen was determined by ELISA with mouse anti-plasminogen antibody. (B) Inhibition of plasminogen binding to fibrinogen by lysin_SM1 (SPR analysis). Lysin_SM1 (1 μM) or PBS was streamed over immobilized fibrinogen followed by plasminogen (0.1 μM). A plot of the level of binding (response units) at equilibrium against a concentration of analyte was used to determine the K_D. For the regeneration studies, lysin_SM1 bound to immobilized fibrinogen was removed by washing with glycine buffer (pH 2.0), streaming with plasminogen (100 nM). (C) Lysin_SM1 expression in the cell wall extract (CW) or culture supernatant (CS) of S. oralis SF100 (WT) and PS1006 (SF100Δlysin) was determined by rabbit anti-lysin_SM1 IgG. (D) Inhibition of plasminogen binding to immobilized fibrinogen by lysin_SM1 from S. oralis SF100. Wells coated with fibrinogen were preincubated with 0 to 100 μl of concentrated (10×) culture supernatant from WT or PS1006, followed by adding plasminogen (0.1 μM). Bound plasminogen was detected with antiplasminogen antibodies. Bars indicate the means (±SD) of triplicate results from a representative experiment. *, = P < 0.05 compared with untreated group.

FIG 6

Inhibition of plasmin(ogen)-mediated fibrinolysis by recombinant lysin_SM1. The impact of lysin_SM1 on fibrin polymerization and proteolysis was assessed by thromboelastography (TEG). Human fibrinogen (2 mg/ml) and 13.7 μM lysin_SM1, lysin_102-198, or lysin_1-101 or albumin (13.7 μM) in HEPES buffer (pH 7.4) were preincubated for 30 min at 37°C. (A and B) Each mixture was transferred to a TEG cup, followed by adding (A) thrombin (1 IU/ml) and plasmin (4 μg/ml) or (B) thrombin (1 IU/ml), tPA (0.5 μg/ml), and plasminogen (4 μg/ml). The clot elastic modulus (n = 2) was recorded once per minute for 30 min. *, P < 0.05 compared with albumin-treated group.

FIG 7

Model of the role of lysin_SM1 during streptococcal proliferation on the surface of a damaged valve. (A) Damaged endocardium becomes covered with platelets and extracellular matrix proteins, including fibrinogen. Thrombin converts fibrinogen to fibrin polymers. (B) Streptococci circulating in the bloodstream bind to fibrin and immobilized platelets on the endovascular surface. Plasminogen binds to the αC region of the fibrinogen Aα chain, followed by recruitment of tPA, which converts plasminogen to plasmin. Plasmin degrades the fibrin clot, thereby disrupting the vegetation and releasing streptococci. (C) Exported lysin_SM1 binds to the αC region of the fibrinogen Aα chain, thereby blocking the binding of plasminogen and inhibiting fibrinolysis.