Figure 1.
Domain organization of lysinSM1 and its homologs.
Domains of lysinSM1 are compared with its closest homologs, the lambdaSa1 phage lysin of Streptococcus agalacitae and the LytA autolysin of Streptococcus pneumoniae, using the NCBI BLAST search program. Levels of identity between regions are indicated. Also shown are the alignments of the amino (N-lysinSM1) and carboxyl terminal (C-lysinSM1) truncated form of lysinSM1. CBD: choline-binding domain. SH3b: putative bacterial cell wall-binding domain.
Figure 2.
Lysin binding to phosphocholine (PC) residue and its biological activity.
(A) Binding of lysinSM1 and its truncated forms to DEAE-cellulose. Samples were separated by SDS-PAGE and stained with Coomassie blue. M: MW standards; lane 1: whole cell extracts of E. coli expressing indicated protein; lane 2: proteins not retained by DEAE cellulose column; lane 3: proteins eluted with 1.5 M NaCl - 0.1% choline chloride; lane 4: proteins eluted with 2% choline chloride. Arrows indicate position of expressed proteins. (B) Binding of FLAGlysinSM1 to immobilized LTA from Streptococcus pneumoniae (SpLTA), PC-negative pneumococci (SpLTA-EA), or Streptococcus mitis (SmLTA). Bars indicate the means (± S.D.) of triplicate results in a representative experiment. (C) Bactericidal activity of lysinSM1. Values shown are number of surviving bacteria after exposure to lysinSM1 or buffer (mean ± S.D.). * = P<0.05, compared with the same strain exposed to buffer alone.
Figure 3.
Lysin binding to platelet monolayers and platelet membranes.
(A) Immobilized human platelets or platelet membranes were incubated with the indicated concentrations of purified FLAGlysinSM1, washed, and bound FLAGlysinSM1 was assessed by ELISA. Bars indicate the means (± S.D.) of triplicate data from a representative experiment. (B) LysinSM1 binding to platelet membrane proteins (far Western blotting). Proteins were either stained with Coomassie blue (panel 1), or transferred to nitrocellulose membranes and probed with FLAGlysinSM1 (panel 2). Platelet membrane proteins were probed with anti-fibrinogen Aα chain (panel 3), anti-fibrinogen Bβ chain (panel 4), or anti-fibrinogen γ chain (panel 5) polyclonal antibodies. (C) Inhibition of FLAGlysinSM1 binding to platelet membranes by fibrinogen IgG polyclonal antibody. Immobilized platelet membranes were incubated with the indicated concentrations of rabbit anti-fibrinogen IgG prior to testing for binding by FLAGlysinSM1 (5 µg/ml). Bars indicate the means (± S.D.) of triplicate results from a representative experiment. * = P<0.05, compared with no antibody treatment.
Figure 4.
(A) Binding of FLAGlysinSM1 to immobilized human fibrinogen. X-axis indicates concentration of FLAGlysinSM1 incubated with fibrinogen. (B) Inhibition of FLAGlysinSM1 binding to immobilized fibrinogen by unlabelled lysinSM1 or fibrinogen. The binding of FLAGlysinSM1 (3 µg/ml) to immobilized fibrinogen was tested in buffer containing 0 to 1,000 µg/ml of lysinSM1 or fibrinogen. * = P<0.05, as compared with 0 µg/ml. (C) Fibrinogen binding to immobilized FLAGlysinSM1, unlabelled lysinSM1, or FLAGAP. Concentrations indicate amount of protein added. Bars are the means (± S.D.) of triplicate results from a representative experiment. (D) Binding of FLAGlysinSM1 to fibrinogen by far Western blotting. Fibrinogen was separated by SDS-PAGE and stained with Coomassie blue (panel 1) or transferred to nitrocellulose and probed with FLAGlysinSM1 (panel 2). The three bands detected in panel 1 correspond to Aα, Bβ, γ chains of fibrinogen. Numbers indicate molecular mass. Binding of FLAGlysinSM1 to the Aα and Bβ chains was readily observed, but not to the γ chain (panel 2).
Figure 5.
Binding of FLAGlysinSM1 to immobilized fibrinogen fragments D and E.
(A) Schematic diagram of human fibrinogen. The Aα, Bβ, and γ chains, major disulfide linkages, and plasmin cleavage sites are shown. (B) Indicated concentrations of FLAGlysinSM1 were incubated with immobilized human fibrinogen, fibrinogen fragment D, or fragment E. Bound FLAGlysinSM1 was detected with anti-FLAG antibody. Bars indicate the means (± S.D.). (C) Fibrinogen fragment D was separated by SDS-PAGE under reducing conditions and stained with Coomassie blue (panel 1) or transferred onto nitrocellulose membrane and incubated with FLAGlysinSM1 (5 µg/ml). The bound proteins were detected with anti-FLAG antibody (Lane 2). Arrows indicate positions of Aα, Bβ, and γ chain fragments. Numbers indicate molecular mass (kDa).
Figure 6.
Lysin mediates binding of bacteria to fibrinogen.
(A) 106 CFU of SF100 or PS1006 were incubated with wells pretreated with the indicated concentrations of fibrinogen. Values represent percent of SF100 binding to wells treated with 300 µg/ml fibrinogen. (B) Complementation of the lys mutant (PS1006) with the lys gene in trans assessed by measuring S. mitis binding to immobilized human fibrinogen. PS1959 (PS1006 complemented with lys) demonstrated significantly greater levels of binding (P<0.05) than PS1006 or PS2093 (PS1006 complemented with pDE123 control vector). Levels of fibrinogen binding by PS1959 were comparable to those seen with SF100 (P>0.05). (C) Binding of FLAGlysinSM1 to PS1006, but not SK598. Bacteria were incubated with purified lysinSM1, and lysinSM1 bound to the cell wall was detected with anti-FLAG antibody. Lane C contains purified lysinSM1 (0.2 µg/ml) as a positive control. (D) PS1006 was incubated with immobilized fibrinogen in the presence of indicated concentration of purified FLAGlysinSM1 (µg/ml). Values represent percent of SF100 binding, and are the means of triplicate results from a representative experiment. * = P<0.05 compared with SF100; # = P>0.05 compared with PS1006.
Table 1.
Impact of lysin expression on virulence in a rat model of infective endocarditis.
Figure 7.
Model for the role of lysin in platelet binding.
Phage lysin is exported through the holin pore and mounted on the bacterial surface of the same or adjacent organisms through its interaction with PC residues. Lysin can then mediate platelet binding via its interaction with fibrinogen and glycoprotein IIb/IIIa (GPIIb/IIIa), which is the principal fibrinogen receptor on platelets. Through its amidase activity, lysin can also permeabilize the cell wall, permitting the release and surface expression PblA and PblB. These phage proteins also interact with platelets by binding the membrane ganglioside GD3.