Characterization of the Fibrinogen Binding Domain of Bacteriophage Lysin from Streptococcus mitis

Abstract

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by the lysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitis to human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysin mediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bind this protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the region spanned by amino acid residues 102 to 198 (lysin_102–198). This region has no sequence homology to other known fibrinogen binding proteins. Lysin_102–198 bound fibrinogen comparably to full-length lysin and with the same selectivity for the fibrinogen Aα and Bβ chains. Lysin_102–198 also inhibited the binding in vitro of S. mitis to human fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene in SF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of the parent strain. The preincubation of platelets with purified lysin_102–198 also delayed the onset of aggregation by SF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of the phage protein and that this interaction is important for both platelet binding and aggregation by S. mitis.

INTRODUCTION

The binding of bacteria to human platelets is thought to be a central event in the pathogenesis of infective endocarditis (14, 61). For numerous endocarditis-associated pathogens, including Staphylococcus aureus, Streptococcus gordonii, and Streptococcus sanguinis, the ability to bind platelets in vitro has been linked to virulence, as measured with animal models of endocarditis (24, 37, 49, 61). Among the viridans group streptococci, Streptococcus mitis is a leading cause of this disease in humans (19, 22, 33). Despite its increasing importance as a pathogen, relatively little is known about the virulence determinants of this organism, particularly with regard to its interaction with platelets or other host components.

Our previous studies found that the lysin encoded by the lysogenic bacteriophage SM1 of S. mitis (lysin_SM1) is expressed on the surface of the bacterium and that this phage protein mediates the binding of S. mitis to human platelets through its interaction with fibrinogen on the platelet surface (30, 47). This appears to be a highly specific interaction, in which lysin binds the Aα and Bβ chains of fibrinogen. Of note, a disruption of the gene encoding lysin (lys) results in both a significant reduction in platelet binding by streptococci in vitro and reduced virulence, indicating that the direct interaction of lysin with fibrinogen contributes to the pathogenesis of endocardial infection by S. mitis.

With a view toward better defining the structural basis of lysin-fibrinogen binding and how this process affects the interaction of S. mitis with platelets, we sought to identify the fibrinogen binding domain of the phage protein. Our studies indicate that a specific domain of lysin_SM1 mediates fibrinogen binding and that this region is important for platelet binding and aggregation by S. mitis.

MATERIALS AND METHODS

Ethics statement.

Blood was obtained from healthy human volunteers, using a protocol approved by the Committee on Human Research at the University of California, San Francisco. All human studies were conducted according to the principles expressed in the Declaration of Helsinki.

Strains and growth conditions.

The bacteria and plasmids used in this study are listed in Table 1. S. mitis and Streptococcus pneumoniae strains were grown in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (THY). S. mitis SF100 is an endocarditis-associated clinical isolate (8, 30). PS1006 is a lys deletion variant of SF100. Both strains grow comparably well in vitro. Escherichia coli strains DH5α and BL21(DE3) were grown at 37°C under aeration in Luria broth (LB; Difco). Appropriate concentrations of antibiotics were added to the medium, as required.

Table 1.

Strains and plasmids

Strain or plasmid	Genotype or descriptiona	Source or reference
Strains
Escherichia coli
DH5α	F− r− m+ φ80dlacZΔM15	Gibco BRL
BL21(DE3)	Expression host; inducible T7 RNA polymerase	Novagen
Streptococcus pneumoniae TIGR4	Serotype 4	ATCC
Streptococcus mitis
SF100	S. mitis; endocarditis clinical isolate	7
PS1006	SF100Δlys::cat Cmr	30
Plasmids
pET28FLAG	Expression vector with 3× FLAG tag	47
pET28FLAGlysinSM1	Vector for expression of 3× FLAG-tagged lysinSM1	47
pET28FLAGlysinSM1	Vector for expression of 3× FLAG-tagged lysinD96E	This study
pMAL-C2X	Expression vector with MalE fusion protein	NEB

^aCm^r, chloramphenicol resistance.

Site-directed mutagenesis.

The replacement of aspartic acid (amino acid [aa] 96) with glutamic acid was done by a two-stage PCR procedure as described previously (56). For codon conversion, either primers 3114-NotI and 5151-D96E or primers 3151-D96E and 5024-XhoI were used to generate to overlapping DNA fragments. The two DNA fragments were then combined for the second-stage PCR and then amplified by using primers 3114-NotI and 5024-XhoI. Amplified products were digested with NotI and XhoI restriction enzymes and ligated into plasmid pET28_FLAG (Table 1).

Cloning and expression of lysin_SM1 and its variants.

Genomic DNA was isolated from SF100 by using Wizard Genomic DNA purification kits (Promega) according to the manufacturer's instructions. PCR was performed with the primers listed in Table 2. To clone truncated lys genes into E. coli expression vectors, PCR products were purified, digested, and ligated into pET28_FLAG to express His-tagged versions of truncated lysins. Cloned plasmids were then introduced to E. coli BL21(DE3) cells by transformation (47). All truncated lysins were purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography (Promega).

Table 2.

Oligonucleotides

Oligonucleotide	Sequence
3114-NotI	GAG CGG CCG CGG GAC TAA ATC TTG
3157-NotI	ACG CGG CCG CCA GCA TGG ATG ACC G
3158-NotI	AAG CGG CCG CTC TAT TGA GTG GAG GAG
3159-NotI	AAG CGG CCG CCT GGC TCA AAA AGA AC
3160-NotI	AAG CGG CCG CTG GAG ATA TCT TCA TTT GG
3161-NotI	AAG CGG CCG CTA TTT TCG TGG ATA GTG ATA AC
3162-NotI	AAG CGG CCG CGA ACG ATC ATG ACG AC
3210-NdeI	AAC ATA TGT TTT CCA TGA GGA TCG TCT GCC
3212-NdeI	AAC ATA TGA AAA GGA TGG TTT CTT GG
3214-SacI	TAG AGC TCG TTG CTA CCA GAG ACA ACT GC
3151-D96E	GAT GCT CAA CGT GGA GAA ATC TTC ATT TGG GGG
5024-XhoI	TAA ACT CGA GTT TCG TTG TGA TC
5160-XhoI	TTC TCG AGA GTG GCC ATG TAG CC
5161-XhoI	TTC TCG AGA TCT TCA GCA ACC ATG
5162-XhoI	TCC TCG AGG TAC TCA AAT TGG TC
5050-XhoI	GCC ACT CGA GTT TGA TTT CTT CGG C
5210-HindIII	TGA AGC TTT CTA GAG GGA AGT AAG TC
5212-XhoI	TCC TCG AGT TTT ACT TGC TTC TGC CTC
5214-XhoI	GTC TCG AGA ACG TCT CCA GCC TGT TTG G
5151-D96E	CCC CCA AAT GAA GAT TTC TCC ACG TTG AGC ATC

Bactericidal assay.

Streptococcus pneumoniae TIGR4 cells were harvested at the early log phase (A₆₀₀ = 0.5) and suspended in phosphate-buffered saline (PBS) at approximately 10⁸ to 10⁹ CFU/ml. Bacterial samples were then incubated with or without 30 μg/ml of purified lysin_SM1 at 37°C, and the change in the optical density (600 nm) was monitored as described previously (40, 47).

Cloning and expression of fibrinogen chains.

cDNAs encoding the Aα, Bβ, and γ chains of human fibrinogen were generously provided by Susan Lord (University of North Carolina at Chapel Hill) (10, 11, 26). Each full-length chain was amplified and cloned into pMAL-C2X (New England BioLabs) to express maltose binding protein (MalE)-tagged versions of the chains. Plasmids were then introduced to E. coli BL21 cells by transformation. All recombinant proteins were purified by affinity chromatography with amylose resin according to the manufacturer's instructions (New England BioLabs).

Analysis of lysin binding to fibrinogen by far-Western blotting.

Purified human fibrinogen and recombinant fibrinogen chains were separated by electrophoresis through 4 to 12% NuPAGE Tris-acetate gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were treated with a casein-based blocking solution (Western blocking reagent; Roche) at room temperature (RT) and then incubated for 1 h with FLAG-tagged lysin_SM1 (_FLAGlysin_SM1) or FLAG-tagged lysin_102–198 (a truncated variant of lysin_SM1 contained within the region spanned by amino acid residues 102 to 198) (1 μM), suspended in PBS–0.05% Tween 20 (PBS-T). The membranes were then washed three times for 15 min in PBS-T, and bound proteins were detected with mouse anti-FLAG antibody (Sigma-Aldrich).

Analysis of lysin binding to fibrinogen by enzyme-linked immunosorbent assay (ELISA).

Purified fibrinogen (10 μg/ml) was immobilized in 96-well microtiter dishes by overnight incubation at 4°C. The wells were washed twice with PBS and blocked with 300 μl of a casein-based blocking solution for 1 h at room temperature. The plates were washed three times with PBS-T, and a range of _FLAGlysin_SM1 or _FLAGlysin_102–198 concentrations in PBS-T was added. The plates were then incubated for 1 h at 37°C. Unbound protein was removed by washing with PBS-T, and the plates were incubated with mouse anti-FLAG antibodies diluted 1:4,000 in PBS-T for 1 h at 37°C. Wells were washed and incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG diluted 1:5,000 in PBS-T for 1 h at 37°C. FLAG-tagged alkaline phosphatase (25 to 100 μg/ml) served as a control for nonspecific binding.

In some studies, fibrinogen was pretreated with a commercial mixture of glycosidases [N-glycanase, sialidase A, O-glycanase, β(1-4)-galactosidase, and β-N-acetylglucosaminidase] according to the manufacturer's instructions (ProZyme). The activity of these enzymes was confirmed by measuring changes in the binding of wheat germ agglutinin (WGA) and succinyl wheat germ agglutinin (sWGA) to immobilized fibrinogen. In brief, after treatment with the glycosidases, the wells were blocked with 300 μl of PBS-T at 37°C for 2 h and washed three times with PBS-T. Purified _FLAGlysin_102–198 or biotinylated WGA (1 μg/ml) and sWGA (1 μg/ml) mixtures were then added, and the plate was incubated at 37°C for 2 h. Unbound antibody and lectins were removed by washing with PBS-T, and bound _FLAGlysin_102–198 or lectins were detected with anti-FLAG monoclonal antibody or streptavidin (1:10,000) in PBS-T as described previously (54).

Analysis of lysin binding to lipoteichoic acid and fibrinogen by ELISA.

Lipoteichoic acid (LTA) was prepared from S. mitis SF100 by organic solvent extraction and octyl-Sepharose chromatography, as previously described (43–46). Purified LTA (1 μg/ml), human fibrinogen (5 μg/ml) (Hematologic Technologies), and recombinant fibrinogen α, β, and γ chains (0.5 μM) in PBS were immobilized in 96-well microtiter dishes by overnight incubation at 4°C. The wells were washed twice with PBS-T and blocked with 300 μl of casein-based blocking solution for 1 h at room temperature. The plates were washed three times with PBS-T, and _FLAGlysin or its truncated variants in PBS-T were added to the wells, over a range of concentrations. The plates were then incubated for 2 h at 37°C. Unbound protein was removed by washing with PBS-T, and plates were incubated with mouse anti-FLAG antibodies for 1 h at 37°C. Binding was assessed by ELISA using HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich).

To examine the relative ability of _FLAGlysin_SM1 or _FLAGlysin_102–198 to inhibit binding to fibrinogen, lysin_SM1 was immobilized in 96-well microtiter plates, followed by the blocking of the wells with the casein blocking solution. Fibrinogen preincubated with _FLAGlysin_SM1 or _FLAGlysin_102–198 over a range of concentrations was added to the wells, and after washing, bound fibrinogen was detected by ELISA using anti-human fibrinogen IgG.

Binding of S. mitis to immobilized platelets and fibrinogen.

Cultures of S. mitis grown overnight were diluted 1:10 in fresh THY broth, incubated for 1 h at 37°C, and then exposed to UV light (λ = 312 nm) for 3 min to induce the expression of the lysogenic bacteriophage SM1 (8, 30). The cultures were then incubated at 37°C for an additional 2 h, followed by harvesting through centrifugation. The pellets were suspended in PBS and adjusted to a concentration of 10⁶ CFU/ml. Purified fibrinogen and human platelets were immobilized in 96-well microtiter plates as described previously (46). The plates were then treated with 300 μl of casein-based blocking solution at 37°C for 1 h and washed three times with PBS. Purified lysin_102–198 (0 to ∼4 μM) was added to the wells for 30 min, followed by the addition of 50 μl of bacteria in PBS. The plates were incubated at room temperature for 1 h, and the wells were washed three times with PBS to remove nonadherent bacteria. The wells were treated with 50 μl of trypsin (2.5 mg/ml) for 10 min at 37°C to release the attached bacteria. The number of bound bacteria was determined by the plating of serial dilutions of the recovered bacteria onto a blood agar plate as previously described (47).

Platelet aggregometry.

The ability of bacteria to induce platelet aggregation was assessed by conventional light aggregometry using a Chronolog model 700 multichannel aggregometer (31). Fifty microliters of bacteria (10⁹ CFU/ml) suspended in Hanks' buffered salt solution was added to 450 μl of stirred human platelet-rich plasma at 37°C. The suspensions were monitored for up to 30 min for the onset of platelet aggregation, as indicated by an increase in light transmission, or until maximal aggregation had occurred. The lag time was defined as the interval between the addition of bacteria to the platelet suspension and the onset of aggregation. Human thrombin (1 μM) or ADP (10 μM) served as a control agonist for normal platelet aggregation. To assess the effect of lysin or its truncated variant lysin_102–198 on aggregation, the platelet-rich plasma was incubated for 5 min with either purified protein over a range of concentrations. Platelet aggregation was then assessed as described above. The statistical significance in lag times was compared by the paired t test.

Quantification of antilysin_SM1 IgG in human sera.

The level of antilysin IgG antibody in human sera was determined by an ELISA (35, 63). In brief, the wells of microtiter plates were treated for 2 h at 37°C with purified lysin_SM1 (1 μg/ml) in PBS, blocked with 300 μl of casein-based solution, and washed three times with PBS-T. Purified lysin_SM1 or lysin_102–198 (0 to 30 μM) was incubated with human sera (1:1,000 diluent) to absorb antilysin antibody for 15 min at RT and added to the wells. Bound IgG was detected with HRP-conjugated rabbit IgG antibody specific for human IgG antibody (Sigma-Aldrich) in PBS-T.

Bioinformatic analysis.

Amino acid homology was compared by using PSI-BLAST, and the secondary structure was predicted by using prediction servers (PHYRE and HHPRED) as described previously (1, 23, 50).

Data analysis.

Data expressed as means ± standard deviations (SD) were compared for statistical significance by the paired or unpaired t test, as indicated.

RESULTS

Identification of the fibrinogen binding domain of lysin.

We have previously observed that lysin encoded by bacteriophage SM1 (lysin_SM1) binds directly to human fibrinogen. To identify the regions within lysin_SM1 involved in this interaction, recombinant forms of the protein containing sequential deletions from the N and C termini were compared for their relative binding to immobilized fibrinogen. When assessed by ELISA, variants of lysin_SM1 containing deletions of 121 amino acids or more from the N terminus (variants 4 and 5) (Fig. 1 A and B) were reduced in binding to fibrinogen compared with the native protein. Lysin variants with shorter deletions (variants 2 and 3) had levels of binding to fibrinogen that were comparable to those of full-length lysin_SM1 (variant 1). The deletion of 126 amino acids or more from the C terminus also abolished the fibrinogen-binding activity of lysin_SM1 (variants 8 and 9), whereas the deletion of 96 or fewer amino acids from this region had no effect on fibrinogen binding (variants 10 and 11). These data suggested that the fibrinogen binding domain of lysin_SM1 is located within aa 102 to 198 of the protein. To confirm these findings, we examined fibrinogen binding by a variant comprised of aa 102 to 198 of lysin_SM1 (lysin_102–198). This polypeptide had levels of fibrinogen binding that were comparable to those of full-length lysin_SM1 (variant 11). In contrast, the deletion of 20 or 40 additional amino acids from the C terminus (variants 12 and 13) resulted in a complete loss of binding, indicating that the entire span of lysin_102–198 was required for fibrinogen binding.

Fig. 1.

Identification of the fibrinogen binding domain of lysin. (A) Schematic of lysin domain organization. The repeating units of the choline binding domain (CBD) are represented by the stripes. Arrows 1 to 13 indicate the truncated forms of lysin tested in these studies. The numbers at the N and C termini indicate the amino acid positions relative to full-length lysin. (B) Binding of lysin and the variants (0.3 μM) to immobilized human fibrinogen (Fg), as measured by ELISA. Full-length lysin served as the standard for 100% binding. Numbers along the x axis correspond to the truncated forms shown in A. (C) Binding of lysin and its truncated variants (0.3 μM) to LTA. Wells coated with fibrinogen or LTA but not incubated with lysin or its variants served as controls. Bars indicate the means (±SD) of triplicate results in a representative experiment.

Bioinformatic analysis of the amino acid sequence of lysin_SM1 revealed that the C terminus contains a predicted choline binding domain (CBD), with six YG repeats (aa 146 to 291) (47). Of note, lysin_102–198 contains two YG repeats of the CBD. To explore the cell wall binding properties of lysin_SM1, we investigated whether the truncated forms of lysin_SM1 bound to immobilized lipoteichoic acid (LTA), a component of the streptococcal cell wall (43, 45) (Fig. 1C). Variants containing N-terminal deletions (variants 2 to 5) showed levels of LTA binding that were comparable to those of native lysin. However, the loss of three or more YG repeats from the C terminus of lysin_SM1 resulted in a marked reduction or complete loss of LTA binding, with lysin_102–198 showing no LTA binding activity. Thus, although lysin_102–198 binds fibrinogen as well as a full-length lysin_SM1, it is insufficient to mediate binding to LTA.

We next assessed the effect of amidase activity on lysin binding to fibrinogen. A lysin variant in which the aspartic acid at residue 96 was replaced with glutamic acid was constructed (lysin_D96E). This substitution is at a highly conserved site within the amidase domains of bacterial and phage lysins that is important for lytic activity (2, 41). The amidase activities of lysin_SM1 and lysin_D96E were measured as bactericidal activity, as described previously (39, 40). As shown in Fig. 2 A, purified lysin_SM1 was found to have highly active bactericidal activity against S. pneumoniae TGR4, producing a reduction of 95.07% ± 5.28% (A₆₀₀). However, no bactericidal activity was seen with lysin_D96E. We then compared the relative binding of _FLAGlysin_D96E to immobilized fibrinogen or LTA with that of _FLAGlysin_SM1 (Fig. 2B and C). The levels of binding of _FLAGlysin_D96E to fibrinogen or LTA were comparable to those of _FLAGlysin_SM1. These data indicate that lytic activity does not contribute to the properties of binding of lysin to either fibrinogen or LTA.

Fig. 2.

Binding of _FLAGlysin and _FLAGlysin_D96E to fibrinogen. (A) Bactericidal activity. Values indicate relative reductions of optical densities (600 nm) after exposure of S. pneumoniae TGR4 to lysin_SM1, lysin_D96E, or PBS alone (means ± SD). (B) Binding of _FLAGlysin or _FLAGlysin_D96E to fibrinogen. Immobilized fibrinogen was incubated with the indicated concentrations of lysins, and relative binding was measured by ELISA. (C) Binding of _FLAGlysin or _FLAGlysin_D96E to immobilized lipoteichoic acid. Bars indicate the means (±SD) of triplicate results in a representative experiment.

Binding of lysin to fibrinogen.

We next compared the binding of immobilized fibrinogen to full-length lysin_SM1 to that of the purified fibrinogen binding domain (lysin_102–198) over a range of concentrations. As shown in Fig. 3 A, _FLAGlysin_102–198 showed levels of fibrinogen binding that were comparable to those seen with full-length _FLAGlysin_SM1. At concentrations above 3 μM, the levels of bound protein reached a plateau for both proteins, indicating that this interaction was becoming saturated.

Fig. 3.

Relative binding of _FLAGlysin and _FLAGlysin_102–198 to fibrinogen. (A) Immobilized fibrinogen was incubated with the indicated concentrations of full-length _FLAGlysin or _FLAGlysin_102–198, and protein binding was assessed by ELISA. Symbols indicate the means (±SD) of triplicate results in a representative experiment. (B) Binding of _FLAGlysin or _FLAGlysin_102–198 to fibrinogen (far-Western blotting). Fibrinogen was separated by SDS-PAGE and stained with Coomassie blue (lanes 1 and 3) or transferred onto nitrocellulose and probed with _FLAGlysin (lane 2) or _FLAGlysin_102–198 (lane 4). The three bands detected in lanes 1 and 3 correspond to the Aα, Bβ, and γ chains of fibrinogen. Binding of _FLAGlysin or _FLAGlysin_102–198 to Aα and Bβ chains was readily observed, with little or no binding to the γ chain. (C) Inhibition of fibrinogen binding to immobilized lysin by _FLAGlysin or _FLAGlysin_102–198. The binding of fibrinogen (1 μg/ml) to immobilized lysin (5 μg/ml) was tested with buffer containing 0 to 30 μM lysin or lysin_102–198. Bound fibrinogen was detected by ELISA. *, P < 0.05, compared with 0 μM. Bars indicate the means (±SD) of triplicate results in a representative experiment.

Fibrinogen is comprised of two subunits, each containing three polypeptide chains (Aα, Bβ, and γ) (25, 32, 58). We have previously shown that full-length lysin_SM1 binds to both the Aα and Bβ chains but minimally binds the γ chain (47). To assess whether _FLAGlysin_102–198 also interacts specifically with these subunits, we examined the binding of _FLAGlysin_102–198 to purified human fibrinogen, as measured by far-Western blotting (Fig. 3B). On SDS-PAGE gels under reducing conditions, fibrinogen appeared as three bands with the expected masses (Aα, 63.5 kDa; Bβ, 56 kDa; γ, 47 kDa). When fibrinogen chains were transferred onto nitrocellulose and probed with _FLAGlysin_SM1 or _FLAGlysin_102–198, both proteins bound predominantly the Aα and Bβ chains. _FLAGlysin_SM1 had very low levels of binding to the γ chain, while no γ chain binding was detected with _FLAGlysin_102–198. As an additional measure of relative binding, we also examined whether fibrinogen binding to immobilized unlabeled lysin_SM1 was blocked by _FLAGlysin_102–198. As shown in Fig. 3C, fibrinogen binding was comparably inhibited by full-length lysin and _FLAGlysin_102–198, indicating that their affinities for the protein were similar.

The binding of some bacterial surface components to human platelets is mediated by the interaction of the adhesin with carbohydrate moieties on their cognate ligand. For example, some members of the serine-rich-repeat family of glycoproteins (such as GspB and Hsa of Streptococcus gordonii and SrpA of Streptococcus sanguinis) bind sialic acid-containing carbohydrates on platelet glycoprotein Ibα (5, 6, 9, 54). To assess the role of carbohydrates in lysin binding to fibrinogen, we treated human fibrinogen with a mixture of glycosidases that remove most N- and simple O-linked carbohydrates from glycoproteins. The treated fibrinogen was then immobilized, and _FLAGlysin_102–198 binding was assessed by ELISA. In control studies, the treatment of fibrinogen with the glycosidases abolished subsequent binding by the lectins WGA and sWGA, indicating that the enzymes were active (data not shown). However, levels of _FLAGlysin_102–198 binding to glycosidase-treated fibrinogen were comparable to those seen with intact fibrinogen (Fig. 4 A).

Fig. 4.

Characterization of fibrinogen binding sites for lysin_102–198. (A) Immobilized glycosylated fibrinogen or deglycosylated fibrinogen (deFg) (5 μg/ml) was incubated with the indicated concentrations of _FLAGlysin_102–198, and bound proteins were assessed by ELISA. (B) Binding of _FLAGlysin_102–198 to recombinant fibrinogen chains. Each fibrinogen chain (expressed as a MalE fusion protein) was probed by far-Western blotting for binding with _FLAGlysin_102–198. (C) Binding of recombinant fibrinogen chains to immobilized lysin_102–198, as measured by ELISA. Symbols indicate the means (±SD) of triplicate results in a representative experiment.

We also assessed the binding of _FLAGlysin_102–198 to recombinant fibrinogen chains purified as MalE fusion proteins from E. coli, an expression background in which these proteins are not glycosylated (Fig. 4B). When separated by SDS-PAGE under reducing conditions, each chain appeared with the expected mass (Aα, 84 kDa; Bβ, 78 kDa; γ, 69 kDa). When probed with _FLAGlysin_102–198, binding to only the Aα and Bβ chains could be detected, confirming the findings seen with purified fibrinogen. Similar results were observed when we assessed the binding of _FLAGlysin_102–198 to recombinant Aα, Bβ, and γ chains by ELISA (Fig. 4C). Purified _FLAGlysin_102–198 (10 μg/ml) was immobilized in microtiter wells and probed with increasing concentrations of recombinant chains in PBS-T. As shown Fig. 4C, no significant binding of the recombinant γ chain to immobilized _FLAGlysin_102–198 was detected. In contrast, recombinant Aα and Bβ chains showed significant binding to immobilized lysin_102–198. These data indicate that, like the full-length protein, lysin_102–198 binds to specific peptide regions on the Aα and Bβ chains of fibrinogen.

Inhibition of S. mitis binding by _FLAGlysin_102–198.

To further confirm the role of the lysin_102–198 region in fibrinogen binding by bacteria, we assessed whether S. mitis binding to immobilized fibrinogen was inhibited by the purified phage protein. In control studies, the parent strain SF100 (wild type [WT]) showed high levels of binding, while its isogenic variant PS1006 (SF100Δlys) had markedly reduced levels of binding, as described previously (Fig. 5 A) (31). When wells containing immobilized fibrinogen were preincubated with purified _FLAGlysin_102–198, the amount of SF100 binding to the wells was subsequently reduced. The level of inhibition was concentration dependent, with 4 μM _FLAGlysin_102–198 being sufficient to reduce SF100 binding to levels comparable to those seen with PS1006.

Fig. 5.

Inhibition of S. mitis binding to fibrinogen and platelets by lysin_102–198. A total of 10⁶ CFU of S. mitis strain SF100 was incubated with immobilized human fibrinogen (A) or platelets (B) in the presence of _FLAGlysin_102–198 at the indicated concentrations (μM). Values represent percentages of SF100 binding and are the means of triplicate results from a representative experiment. Strain PS1006 (SF100Δlys) served as a control for low binding.

We then examined whether purified lysin_102–198 could inhibit bacterial binding to human platelets. SF100 demonstrated high levels of binding to immobilized platelets, as described above. The preincubation of the platelet monolayers with purified _FLAGlysin_102–198 significantly reduced bacterial binding to immobilized platelets, with the level of inhibition being proportional to the amount of protein added (Fig. 5B). In control studies, _FLAGlysin_102–198 had no effect on the low levels of fibrinogen or platelet binding seen with PS1006 (data not shown). These data further indicate that SF100 binding to fibrinogen on platelet membranes is mediated by aa 102 to 198 of lysin_SM1.

Role of lysin in platelet aggregation.

To assess further the effect of lysin on the interaction of bacteria with platelets, we tested SF100 and PS1006 for the ability to induce platelet aggregation (Fig. 6). Both strains produced comparable levels of aggregation in vitro, as measured by the maximum change in light transmission (Fig. 6A). However, the time to the onset of aggregation was significantly longer for PS1006 (18.0 ± 8.6 min [mean ± SD]) than for SF100 (12.1 ± 6.6 min; P = 0.03) (Fig. 6C). We then tested whether the preincubation of platelets with _FLAGlysin_102–198 affected subsequent aggregation by SF100 (Fig. 6B and C). We found that _FLAGlysin_102–198 inhibited platelet aggregation by SF100 in a concentration-dependent manner, with as little as 4 nM polypeptide resulting in a significant prolongation of the lag time. At 4 μM, _FLAGlysin_102–198 completely blocked the aggregation of platelets from all four donors tested. In control studies, lysin_102–198 had no effect on aggregation by PS1006 (data not shown).

Fig. 6.

Effect of lysin_SM1 on platelet aggregation by bacteria. (A) Strain SF100 or PS1006 was added at 0 min to stirred platelet-rich plasma, and the suspensions were monitored for changes in light transmission. Representative data from a single donor are shown. (B) Platelets were preincubated with the indicated concentrations of lysin_102–198 followed by the addition of SF100. Representative data from a single donor are shown. (C) Effect of lysin on the time to the onset of platelet aggregation (lag time). The table shows results using platelets obtained from four different donors.

The above-described studies indicated that the binding of lysin to fibrinogen contributes to the induction of platelet aggregation by SF100. However, platelet aggregation by most bacteria, including SF100, requires IgG specific for the organism, which, after binding to the bacterial surface, triggers activation through the platelet FcγRII receptor (30, 51–53; data not shown). It was conceivable, therefore, that the prolonged lag times described above were due to the adsorption of antilysin antibodies by soluble _FLAGlysin_102–198, thereby reducing the amount of IgG available for bacterial binding and FcγRII signaling. To assess this possibility, we tested sera from several human donors for the presence of antilysin_SM1 IgG (Fig. 7). When examined by ELISA, high levels of antilysin_SM1 IgG were present in all donor sera (n = 4). Antibodies to lysin_102–198 were also detected but at considerably lower levels (Fig. 7A). In addition, the binding of IgG to immobilized lysin_SM1 was inhibited by preincubating the sera with purified lysin_SM1 but not with lysin_102–198 (Fig. 7B). These findings indicate that the inhibition of platelet aggregation by lysin_102–198 is unlikely to be due to the adsorption of lysin-specific IgG and reduced FcγRII-mediated activation. Rather, the prolonged lag times seen with the pretreatment of platelets with lysin_102–198 is more likely to due to the inhibition of streptococcal binding to fibrinogen on the platelet surface, resulting in a delay in the onset of aggregation.

Fig. 7.

Levels of antilysin IgG in human sera and inhibition of antibody binding to immobilized lysin_SM1 by soluble lysin_SM1 or lysin_102–198. (A) Immobilized lysin_SM1 or lysin_102–198 was incubated with diluted sera from four donors, and the amount of IgG bound was measured by ELISA. (B) Sera from four donors were mixed with the indicated concentrations (μM) of lysin_SM1 or lysin_102–198 and then tested individually for antibody binding to immobilized lysin_SM1 by ELISA.

DISCUSSION

The pathogenesis of endocarditis is a multistep process comprised of numerous host-pathogen interactions (14). Infection of the endocardium is typically initiated by the attachment of blood-borne organisms to damaged or prosthetic cardiac valve surfaces that are covered by platelets and plasma proteins (34, 59). This binding event is rapidly followed by the subsequent further deposition of platelets and other proteins, which, in conjunction with microbial proliferation, results in the formation of macroscopic vegetations.

Our previous studies have identified several surface proteins of S. mitis mediating binding to human platelets that are encoded by a lysogenic bacteriophage, SM1 (30, 47). These proteins (PblA, PblB, and lysin_SM1) are expressed during the phage lytic cycle and attach to the bacterial cell surface via the interaction of their choline binding domains with lipoteichoic acid. The expression of these proteins by S. mitis is associated with increased virulence in animal models of endocarditis. Of note, recent studies indicated that homologs of PblA, PblB, and lysin are common among a range of Gram-positive pathogens and that SM1 is a ubiquitous member of the oral microbiota (3, 42, 60, 62). Since oral streptococci are among the most frequent causes of infective endocarditis, it is likely that these phage-encoded adhesins are highly conserved virulence factors.

We recently found that lysin_SM1 binds platelets in vitro through its interaction with fibrinogen on the platelet surface (47). However, it was unknown which region of the phage protein mediated this interaction, and because lysin_SM1 lacks homology with other known fibrinogen binding proteins, no candidate regions were apparent. We now report that the region of lysin_SM1 corresponding to residues 102 to 198 mediates this interaction and that lysin_102–198 has fibrinogen binding properties that are comparable to those of the full-length protein. In particular, the two proteins appeared to have comparable affinities for fibrinogen. Both full-length lysin and lysin_102–198 selectively bound the Aα and Bβ chains of fibrinogen, and the binding of lysin_SM1 was effectively blocked by the polypeptide. These results indicate that the fibrinogen binding properties of lysin are contained wholly within this domain.

A large number of fibrinogen binding proteins of Gram-positive bacteria have been identified in recent years, such as the ClfA, FnbpA, SdrG, and Aaa proteins of Staphylococcus and the M proteins of Streptococcus pyogenes (12, 13, 15, 20, 21, 57). Among the best characterized of the staphylococcal fibrinogen binding proteins are ClfA and SdrG, where the solution of their crystal structures in complex with fibrinogen-derived peptides has revealed the structural basis for binding. Both ClfA and SdrG contain two Ig-like domains that form a pocket for fibrinogen binding via a “dock, lock, and latch” mechanism (38). These proteins differ, however, in that ClfA recognizes the C terminus of the γ chain, whereas SdrG recognizes the N terminus of the β chain (17, 38). The crystal structure of the M1 protein of S. pyogenes in complex with fibrinogen fragment D was reported recently (28). These proteins were found to form a cross-like complex comprised of a coiled-coil dimer of M1 bound to four molecules of fragment D. Less is known about the details of fibrinogen binding by other bacterial proteins, due to the lack of structural information. Proteins such as Eap and Efb have been shown to bind the Aα chain through a series of tandem repeats, but how these subdomains form a complex with fibrinogen has not been determined (12, 18, 55).

Although the precise structural requirements for fibrinogen binding by lysin_SM1 are not fully known, the predicted amino acid sequence of lysin_102–198 has no significant homology to any of the above-described fibrinogen binding proteins. In addition, structure prediction searches (using PSI-BLAST, PHYRE, and HHPRED) (1, 23, 50) failed to identify any potential structural similarities between lysin_SM1 and these fibrinogen binding proteins (data not shown), further indicating that lysin_SM1 binds fibrinogen by a distinct mechanism. As for other bacterial proteins known to bind fibrinogen, one possible homolog of lysin_SM1 is choline binding protein E (CbpE) of S. pneumoniae. CbpE is a cell wall-associated protein and a member of the metallo-β-lactamase superfamily that has been shown to bind fibrinogen in vitro (16). Although the structure of CbpE has been solved, the regions of this proteins involved in fibrinogen binding have not been identified. CbpE shares only 20% overall amino acid identity with lysin_SM1, and an alignment of the proteins indicates that the region of CbpE corresponding to lysin_102–198 has even less identity (17%). However, secondary-structure analysis predicts that these proteins share a high level of similarity in folding, suggesting that they could bind fibrinogen by a similar mechanism.

In addition to mediating platelet binding, we found that phage lysin contributes to platelet aggregation by S. mitis. The loss of lysin expression was associated with a prolonged lag time, which also reflects the time to the onset of platelet activation (15, 27, 29, 34, 36). The preincubation of platelets with lysin_102–198 prior to mixing with whole bacteria also delayed aggregation, further indicating that fibrinogen binding by lysin contributes to aggregation. These findings are consistent with previous studies of several staphylococcal proteins (e.g., ClfA, ClfB, and FnBpA), which were also found to promote platelet aggregation via their interactions with fibrinogen (4, 29, 34, 48). Of note, platelet aggregation by SF100 (and most bacteria) requires IgG specific for the organism, which, once bound to the bacterial surface, interacts with the platelet FcγRII to induce activation and aggregation (14, 15, 27). This finding raised the possibility that the effect of lysin on the lag time may have been due to its interaction with specific antibodies and the engagement of FcγRII, rather than fibrinogen binding. However, although sera from our donors did contain antibodies to full-length lysin, only a minor component appeared to be directed against the fibrinogen binding domain. It is thus unlikely that antibodies specific for this domain contribute significantly to activation. In contrast, since even nanomolar concentrations of lysin_102–198 in solution prolonged the lag time, it is likely that the lysin on the bacterial surface accelerates platelet aggregation through its interaction with fibrinogen.

In summary, the lysin of bacteriophage SM1 appears to be a novel fibrinogen binding protein. The binding region of lysin_SM1 has no predicted structural similarities to previously characterized binding domains of other fibrinogen binding proteins. Thus, the precise basis for lysin binding and the promotion of aggregation is unknown and will require additional structural analyses, which are now in progress.