Streptococcus mitis

INFECTION AND IMMUNITY, Sept. 2011, p. 3518–3526 Vol. 79, No. 90019-9567/11/$12.00 doi:10.1128/IAI.05088-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Characterization of the Fibrinogen Binding Domain ofBacteriophage Lysin from Streptococcus mitis�

Ho Seong Seo and Paul M. Sullam*Division of Infectious Diseases, Veterans Affairs Medical Center and the University of California, San Francisco, California

Received 18 March 2011/Returned for modification 12 April 2011/Accepted 9 June 2011

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infectiveendocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by thelysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitisto human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysinmediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bindthis protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the regionspanned by amino acid residues 102 to 198 (lysin102–198). This region has no sequence homology to other knownfibrinogen binding proteins. Lysin102–198 bound fibrinogen comparably to full-length lysin and with the sameselectivity for the fibrinogen A� and B� chains. Lysin102–198 also inhibited the binding in vitro of S. mitis tohuman fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene inSF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of theparent strain. The preincubation of platelets with purified lysin102–198 also delayed the onset of aggregation bySF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of thephage protein and that this interaction is important for both platelet binding and aggregation by S. mitis.

The binding of bacteria to human platelets is thought to bea central event in the pathogenesis of infective endocarditis(14, 61). For numerous endocarditis-associated pathogens, in-cluding Staphylococcus aureus, Streptococcus gordonii, andStreptococcus sanguinis, the ability to bind platelets in vitro hasbeen linked to virulence, as measured with animal models ofendocarditis (24, 37, 49, 61). Among the viridans group strep-tococci, Streptococcus mitis is a leading cause of this disease inhumans (19, 22, 33). Despite its increasing importance as apathogen, relatively little is known about the virulence deter-minants of this organism, particularly with regard to its inter-action with platelets or other host components.

Our previous studies found that the lysin encoded by thelysogenic bacteriophage SM1 of S. mitis (lysinSM1) is expressedon the surface of the bacterium and that this phage proteinmediates the binding of S. mitis to human platelets through itsinteraction with fibrinogen on the platelet surface (30, 47).This appears to be a highly specific interaction, in which lysinbinds the A� and B� chains of fibrinogen. Of note, a disruptionof the gene encoding lysin (lys) results in both a significantreduction in platelet binding by streptococci in vitro and re-duced virulence, indicating that the direct interaction of lysinwith fibrinogen contributes to the pathogenesis of endocardialinfection by S. mitis.

With a view toward better defining the structural basis oflysin-fibrinogen binding and how this process affects the inter-action of S. mitis with platelets, we sought to identify thefibrinogen binding domain of the phage protein. Our studiesindicate that a specific domain of lysinSM1 mediates fibrinogen

binding and that this region is important for platelet bindingand aggregation by S. mitis.

MATERIALS AND METHODS

Ethics statement. Blood was obtained from healthy human volunteers, using aprotocol approved by the Committee on Human Research at the University ofCalifornia, San Francisco. All human studies were conducted according to theprinciples expressed in the Declaration of Helsinki.

Strains and growth conditions. The bacteria and plasmids used in this studyare listed in Table 1. S. mitis and Streptococcus pneumoniae strains were grownin 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 lysdeletion variant of SF100. Both strains grow comparably well in vitro. Escherichiacoli strains DH5� and BL21(DE3) were grown at 37°C under aeration in Luriabroth (LB; Difco). Appropriate concentrations of antibiotics were added to themedium, as required.

Site-directed mutagenesis. The replacement of aspartic acid (amino acid [aa]96) with glutamic acid was done by a two-stage PCR procedure as describedpreviously (56). For codon conversion, either primers 3114-NotI and 5151-D96Eor primers 3151-D96E and 5024-XhoI were used to generate to overlappingDNA 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 andligated into plasmid pET28FLAG (Table 1).

Cloning and expression of lysinSM1 and its variants. Genomic DNA wasisolated from SF100 by using Wizard Genomic DNA purification kits (Promega)according to the manufacturer’s instructions. PCR was performed with the prim-ers listed in Table 2. To clone truncated lys genes into E. coli expression vectors,PCR products were purified, digested, and ligated into pET28FLAG to expressHis-tagged versions of truncated lysins. Cloned plasmids were then introduced toE. coli BL21(DE3) cells by transformation (47). All truncated lysins were purifiedby Ni-nitrilotriacetic acid (NTA) affinity chromatography (Promega).

Bactericidal assay. Streptococcus pneumoniae TIGR4 cells were harvested atthe early log phase (A600 � 0.5) and suspended in phosphate-buffered saline(PBS) at approximately 108 to 109 CFU/ml. Bacterial samples were then incu-bated with or without 30 �g/ml of purified lysinSM1 at 37°C, and the change in theoptical density (600 nm) was monitored as described previously (40, 47).

Cloning and expression of fibrinogen chains. cDNAs encoding the A�, B�,and � chains of human fibrinogen were generously provided by Susan Lord(University of North Carolina at Chapel Hill) (10, 11, 26). Each full-length chainwas amplified and cloned into pMAL-C2X (New England BioLabs) to express

* Corresponding author. Mailing address: Division of InfectiousDiseases, VA Medical Center (111W), 4150 Clement St., San Fran-cisco, CA 94121. Phone: (415) 221-4810, ext. 2550. Fax: (415) 750-6951. E-mail: [email protected].

� Published ahead of print on 20 June 2011.

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maltose binding protein (MalE)-tagged versions of the chains. Plasmids werethen introduced to E. coli BL21 cells by transformation. All recombinant proteinswere purified by affinity chromatography with amylose resin according to themanufacturer’s instructions (New England BioLabs).

Analysis of lysin binding to fibrinogen by far-Western blotting. Purified hu-man fibrinogen and recombinant fibrinogen chains were separated by electro-phoresis through 4 to 12% NuPAGE Tris-acetate gels (Invitrogen) and trans-ferred onto nitrocellulose membranes. The membranes were treated with acasein-based blocking solution (Western blocking reagent; Roche) at roomtemperature (RT) and then incubated for 1 h with FLAG-tagged lysinSM1

(FLAGlysinSM1) or FLAG-tagged lysin102–198 (a truncated variant of lysinSM1

contained within the region spanned by amino acid residues 102 to 198) (1 �M),suspended in PBS–0.05% Tween 20 (PBS-T). The membranes were then washedthree times for 15 min in PBS-T, and bound proteins were detected with mouseanti-FLAG antibody (Sigma-Aldrich).

Analysis of lysin binding to fibrinogen by enzyme-linked immunosorbent as-say (ELISA). Purified fibrinogen (10 �g/ml) was immobilized in 96-well micro-titer dishes by overnight incubation at 4°C. The wells were washed twice withPBS and blocked with 300 �l of a casein-based blocking solution for 1 h at roomtemperature. The plates were washed three times with PBS-T, and a range ofFLAGlysinSM1 or FLAGlysin102–198 concentrations in PBS-T was added. The plateswere then incubated for 1 h at 37°C. Unbound protein was removed by washingwith PBS-T, and the plates were incubated with mouse anti-FLAG antibodiesdiluted 1:4,000 in PBS-T for 1 h at 37°C. Wells were washed and incubated withhorseradish 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 �g/ml)served as a control for nonspecific binding.

In some studies, fibrinogen was pretreated with a commercial mixture ofglycosidases [N-glycanase, sialidase A, O-glycanase, �(1-4)-galactosidase, and�-N-acetylglucosaminidase] according to the manufacturer’s instructions(ProZyme). The activity of these enzymes was confirmed by measuring changesin the binding of wheat germ agglutinin (WGA) and succinyl wheat germ agglu-tinin (sWGA) to immobilized fibrinogen. In brief, after treatment with theglycosidases, the wells were blocked with 300 �l of PBS-T at 37°C for 2 h andwashed three times with PBS-T. Purified FLAGlysin102–198 or biotinylated WGA(1 �g/ml) and sWGA (1 �g/ml) mixtures were then added, and the plate wasincubated at 37°C for 2 h. Unbound antibody and lectins were removed bywashing with PBS-T, and bound FLAGlysin102–198 or lectins were detected withanti-FLAG monoclonal antibody or streptavidin (1:10,000) in PBS-T as de-scribed previously (54).

Analysis of lysin binding to lipoteichoic acid and fibrinogen by ELISA. Lipo-teichoic acid (LTA) was prepared from S. mitis SF100 by organic solvent extrac-tion and octyl-Sepharose chromatography, as previously described (43–46). Pu-rified LTA (1 �g/ml), human fibrinogen (5 �g/ml) (Hematologic Technologies),and recombinant fibrinogen �, �, and � chains (0.5 �M) in PBS were immobi-lized in 96-well microtiter dishes by overnight incubation at 4°C. The wells werewashed twice with PBS-T and blocked with 300 �l of casein-based blockingsolution for 1 h at room temperature. The plates were washed three times withPBS-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.

TABLE 1. Strains and plasmids

Strain or plasmid Genotype or descriptiona Source or reference

StrainsEscherichia coli

DH5� F� r� m� �80dlacZM15 Gibco BRLBL21(DE3) Expression host; inducible T7 RNA polymerase Novagen

Streptococcus pneumoniae TIGR4 Serotype 4 ATCCStreptococcus mitis

SF100 S. mitis; endocarditis clinical isolate 7PS1006 SF100lys::cat Cmr 30

PlasmidspET28FLAG Expression vector with 3 FLAG tag 47pET28FLAGlysinSM1 Vector for expression of 3 FLAG-tagged lysinSM1 47pET28FLAGlysinSM1 Vector for expression of 3 FLAG-tagged lysinD96E This studypMAL-C2X Expression vector with MalE fusion protein NEB

a Cmr, chloramphenicol resistance.

TABLE 2. Oligonucleotides

Oligonucleotide Sequence

3114-NotI.......................................................................................................................GAG CGG CCG CGG GAC TAA ATC TTG3157-NotI.......................................................................................................................ACG CGG CCG CCA GCA TGG ATG ACC G3158-NotI.......................................................................................................................AAG CGG CCG CTC TAT TGA GTG GAG GAG3159-NotI.......................................................................................................................AAG CGG CCG CCT GGC TCA AAA AGA AC3160-NotI.......................................................................................................................AAG CGG CCG CTG GAG ATA TCT TCA TTT GG3161-NotI.......................................................................................................................AAG CGG CCG CTA TTT TCG TGG ATA GTG ATA AC3162-NotI.......................................................................................................................AAG CGG CCG CGA ACG ATC ATG ACG AC3210-NdeI ......................................................................................................................AAC ATA TGT TTT CCA TGA GGA TCG TCT GCC3212-NdeI ......................................................................................................................AAC ATA TGA AAA GGA TGG TTT CTT GG3214-SacI .......................................................................................................................TAG AGC TCG TTG CTA CCA GAG ACA ACT GC3151-D96E.....................................................................................................................GAT GCT CAA CGT GGA GAA ATC TTC ATT TGG GGG5024-XhoI......................................................................................................................TAA ACT CGA GTT TCG TTG TGA TC5160-XhoI......................................................................................................................TTC TCG AGA GTG GCC ATG TAG CC5161-XhoI......................................................................................................................TTC TCG AGA TCT TCA GCA ACC ATG5162-XhoI......................................................................................................................TCC TCG AGG TAC TCA AAT TGG TC5050-XhoI......................................................................................................................GCC ACT CGA GTT TGA TTT CTT CGG C5210-HindIII .................................................................................................................TGA AGC TTT CTA GAG GGA AGT AAG TC5212-XhoI......................................................................................................................TCC TCG AGT TTT ACT TGC TTC TGC CTC5214-XhoI......................................................................................................................GTC TCG AGA ACG TCT CCA GCC TGT TTG G5151-D96E.....................................................................................................................CCC CCA AAT GAA GAT TTC TCC ACG TTG AGC ATC

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Unbound protein was removed by washing with PBS-T, and plates were incu-bated with mouse anti-FLAG antibodies for 1 h at 37°C. Binding was assessed byELISA using HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich).

To examine the relative ability of FLAGlysinSM1 or FLAGlysin102–198 to inhibitbinding to fibrinogen, lysinSM1 was immobilized in 96-well microtiter plates,followed by the blocking of the wells with the casein blocking solution. Fibrin-ogen preincubated with FLAGlysinSM1 or FLAGlysin102–198 over a range of con-centrations was added to the wells, and after washing, bound fibrinogen wasdetected by ELISA using anti-human fibrinogen IgG.

Binding of S. mitis to immobilized platelets and fibrinogen. Cultures of S. mitisgrown overnight were diluted 1:10 in fresh THY broth, incubated for 1 h at 37°C,and then exposed to UV light (� � 312 nm) for 3 min to induce the expressionof the lysogenic bacteriophage SM1 (8, 30). The cultures were then incubated at37°C for an additional 2 h, followed by harvesting through centrifugation. Thepellets were suspended in PBS and adjusted to a concentration of 106 CFU/ml.Purified fibrinogen and human platelets were immobilized in 96-well microtiterplates as described previously (46). The plates were then treated with 300 �l ofcasein-based blocking solution at 37°C for 1 h and washed three times with PBS.Purified lysin102–198 (0 to �4 �M) was added to the wells for 30 min, followed bythe addition of 50 �l of bacteria in PBS. The plates were incubated at roomtemperature for 1 h, and the wells were washed three times with PBS to removenonadherent bacteria. The wells were treated with 50 �l of trypsin (2.5 mg/ml)for 10 min at 37°C to release the attached bacteria. The number of boundbacteria was determined by the plating of serial dilutions of the recoveredbacteria onto a blood agar plate as previously described (47).

Platelet aggregometry. The ability of bacteria to induce platelet aggregationwas assessed by conventional light aggregometry using a Chronolog model 700multichannel aggregometer (31). Fifty microliters of bacteria (109 CFU/ml)suspended in Hanks’ buffered salt solution was added to 450 �l of stirred humanplatelet-rich plasma at 37°C. The suspensions were monitored for up to 30 minfor the onset of platelet aggregation, as indicated by an increase in light trans-mission, or until maximal aggregation had occurred. The lag time was defined asthe interval between the addition of bacteria to the platelet suspension and theonset of aggregation. Human thrombin (1 �M) or ADP (10 �M) served as acontrol agonist for normal platelet aggregation. To assess the effect of lysin or itstruncated variant lysin102–198 on aggregation, the platelet-rich plasma was incu-bated for 5 min with either purified protein over a range of concentrations.Platelet aggregation was then assessed as described above. The statistical signif-icance in lag times was compared by the paired t test.

Quantification of antilysinSM1 IgG in human sera. The level of antilysin IgGantibody in human sera was determined by an ELISA (35, 63). In brief, the wellsof microtiter plates were treated for 2 h at 37°C with purified lysinSM1 (1 �g/ml)in PBS, blocked with 300 �l of casein-based solution, and washed three timeswith PBS-T. Purified lysinSM1 or lysin102–198 (0 to 30 �M) was incubated withhuman sera (1:1,000 diluent) to absorb antilysin antibody for 15 min at RT andadded to the wells. Bound IgG was detected with HRP-conjugated rabbit IgGantibody 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) werecompared for statistical significance by the paired or unpaired t test, as indicated.

RESULTS

Identification of the fibrinogen binding domain of lysin. Wehave previously observed that lysin encoded by bacteriophageSM1 (lysinSM1) binds directly to human fibrinogen. To identifythe regions within lysinSM1 involved in this interaction, recom-binant forms of the protein containing sequential deletionsfrom the N and C termini were compared for their relativebinding to immobilized fibrinogen. When assessed by ELISA,variants of lysinSM1 containing deletions of 121 amino acids ormore from the N terminus (variants 4 and 5) (Fig. 1A and B)were reduced in binding to fibrinogen compared with the na-tive protein. Lysin variants with shorter deletions (variants 2and 3) had levels of binding to fibrinogen that were compara-ble to those of full-length lysinSM1 (variant 1). The deletion of126 amino acids or more from the C terminus also abolishedthe fibrinogen-binding activity of lysinSM1 (variants 8 and 9),whereas the deletion of 96 or fewer amino acids from thisregion had no effect on fibrinogen binding (variants 10 and 11).These data suggested that the fibrinogen binding domain of

FIG. 1. Identification of the fibrinogen binding domain of lysin. (A) Schematic of lysin domain organization. The repeating units of the cholinebinding domain (CBD) are represented by the stripes. Arrows 1 to 13 indicate the truncated forms of lysin tested in these studies. The numbersat the N and C termini indicate the amino acid positions relative to full-length lysin. (B) Binding of lysin and the variants (0.3 �M) to immobilizedhuman fibrinogen (Fg), as measured by ELISA. Full-length lysin served as the standard for 100% binding. Numbers along the x axis correspondto the truncated forms shown in A. (C) Binding of lysin and its truncated variants (0.3 �M) to LTA. Wells coated with fibrinogen or LTA but notincubated with lysin or its variants served as controls. Bars indicate the means ( SD) of triplicate results in a representative experiment.

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lysinSM1 is located within aa 102 to 198 of the protein. Toconfirm these findings, we examined fibrinogen binding by avariant comprised of aa 102 to 198 of lysinSM1 (lysin102–198).This polypeptide had levels of fibrinogen binding that werecomparable to those of full-length lysinSM1 (variant 11). Incontrast, the deletion of 20 or 40 additional amino acids fromthe C terminus (variants 12 and 13) resulted in a complete lossof binding, indicating that the entire span of lysin102–198 was re-quired for fibrinogen binding.

Bioinformatic analysis of the amino acid sequence oflysinSM1 revealed that the C terminus contains a predictedcholine binding domain (CBD), with six YG repeats (aa 146to 291) (47). Of note, lysin102–198 contains two YG repeatsof the CBD. To explore the cell wall binding properties oflysinSM1, we investigated whether the truncated forms oflysinSM1 bound to immobilized lipoteichoic acid (LTA), acomponent 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 tothose of native lysin. However, the loss of three or more YGrepeats from the C terminus of lysinSM1 resulted in amarked reduction or complete loss of LTA binding, withlysin102–198 showing no LTA binding activity. Thus, althoughlysin102–198 binds fibrinogen as well as a full-length lysinSM1,it is insufficient to mediate binding to LTA.

We next assessed the effect of amidase activity on lysinbinding to fibrinogen. A lysin variant in which the aspartic acidat residue 96 was replaced with glutamic acid was constructed(lysinD96E). This substitution is at a highly conserved sitewithin the amidase domains of bacterial and phage lysins thatis important for lytic activity (2, 41). The amidase activities oflysinSM1 and lysinD96E were measured as bactericidal activity,as described previously (39, 40). As shown in Fig. 2A, purifiedlysinSM1 was found to have highly active bactericidal activityagainst S. pneumoniae TGR4, producing a reduction of95.07% 5.28% (A600). However, no bactericidal activity wasseen with lysinD96E. We then compared the relative binding ofFLAGlysinD96E to immobilized fibrinogen or LTA with thatof FLAGlysinSM1 (Fig. 2B and C). The levels of binding ofFLAGlysinD96E to fibrinogen or LTA were comparable to thoseof FLAGlysinSM1. These data indicate that lytic activity does not

contribute to the properties of binding of lysin to either fibrin-ogen or LTA.

Binding of lysin to fibrinogen. We next compared the bind-ing of immobilized fibrinogen to full-length lysinSM1 to that ofthe purified fibrinogen binding domain (lysin102–198) over arange of concentrations. As shown in Fig. 3A, FLAGlysin102–198

showed levels of fibrinogen binding that were comparable tothose seen with full-length FLAGlysinSM1. At concentrationsabove 3 �M, the levels of bound protein reached a plateau forboth proteins, indicating that this interaction was becomingsaturated.

Fibrinogen is comprised of two subunits, each containingthree polypeptide chains (A�, B�, and �) (25, 32, 58). We havepreviously shown that full-length lysinSM1 binds to both the A�and B� chains but minimally binds the � chain (47). To assesswhether FLAGlysin102–198 also interacts specifically with thesesubunits, we examined the binding of FLAGlysin102–198 to puri-fied 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(A�, 63.5 kDa; B�, 56 kDa; �, 47 kDa). When fibrinogenchains were transferred onto nitrocellulose and probed withFLAGlysinSM1 or FLAGlysin102–198, both proteins bound predom-inantly the A� and B� chains. FLAGlysinSM1 had very low levels ofbinding to the � chain, while no � chain binding was detected withFLAGlysin102–198. As an additional measure of relative binding, wealso examined whether fibrinogen binding to immobilized unla-beled lysinSM1 was blocked by FLAGlysin102–198. As shown in Fig.3C, fibrinogen binding was comparably inhibited by full-lengthlysin and FLAGlysin102–198, indicating that their affinities for theprotein were similar.

The binding of some bacterial surface components to humanplatelets is mediated by the interaction of the adhesin withcarbohydrate moieties on their cognate ligand. For example,some members of the serine-rich-repeat family of glycopro-teins (such as GspB and Hsa of Streptococcus gordonii andSrpA of Streptococcus sanguinis) bind sialic acid-containingcarbohydrates on platelet glycoprotein Ib� (5, 6, 9, 54). Toassess the role of carbohydrates in lysin binding to fibrinogen,we treated human fibrinogen with a mixture of glycosidasesthat remove most N- and simple O-linked carbohydrates from

FIG. 2. Binding of FLAGlysin and FLAGlysinD96E to fibrinogen. (A) Bactericidal activity. Values indicate relative reductions of optical densities(600 nm) after exposure of S. pneumoniae TGR4 to lysinSM1, lysinD96E, or PBS alone (means SD). (B) Binding of FLAGlysin or FLAGlysinD96Eto fibrinogen. Immobilized fibrinogen was incubated with the indicated concentrations of lysins, and relative binding was measured by ELISA.(C) Binding of FLAGlysin or FLAGlysinD96E to immobilized lipoteichoic acid. Bars indicate the means ( SD) of triplicate results in a representativeexperiment.



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glycoproteins. The treated fibrinogen was then immobilized,and FLAGlysin102–198 binding was assessed by ELISA. In con-trol studies, the treatment of fibrinogen with the glycosidasesabolished subsequent binding by the lectins WGA and sWGA,indicating that the enzymes were active (data not shown).However, levels of FLAGlysin102–198 binding to glycosidase-treated fibrinogen were comparable to those seen with intactfibrinogen (Fig. 4A).

We also assessed the binding of FLAGlysin102–198 to recom-binant fibrinogen chains purified as MalE fusion proteins fromE. coli, an expression background in which these proteins arenot glycosylated (Fig. 4B). When separated by SDS-PAGEunder reducing conditions, each chain appeared with the ex-pected mass (A�, 84 kDa; B�, 78 kDa; �, 69 kDa). Whenprobed with FLAGlysin102–198, binding to only the A� and B�chains could be detected, confirming the findings seen withpurified fibrinogen. Similar results were observed when we

assessed the binding of FLAGlysin102–198 to recombinant A�,B�, and � chains by ELISA (Fig. 4C). Purified FLAGlysin102–198

(10 �g/ml) was immobilized in microtiter wells and probedwith increasing concentrations of recombinant chains inPBS-T. As shown Fig. 4C, no significant binding of the recom-binant � chain to immobilized FLAGlysin102–198 was detected.In contrast, recombinant A� and B� chains showed significantbinding to immobilized lysin102–198. These data indicate that,like the full-length protein, lysin102–198 binds to specific peptideregions on the A� and B� chains of fibrinogen.

Inhibition of S. mitis binding by FLAGlysin102–198. To furtherconfirm the role of the lysin102–198 region in fibrinogen bindingby bacteria, we assessed whether S. mitis binding to immobi-lized fibrinogen was inhibited by the purified phage protein. Incontrol studies, the parent strain SF100 (wild type [WT]) showedhigh levels of binding, while its isogenic variant PS1006(SF100lys) had markedly reduced levels of binding, as described

FIG. 3. Relative binding of FLAGlysin and FLAGlysin102–198 to fibrinogen. (A) Immobilized fibrinogen was incubated with the indicatedconcentrations of full-length FLAGlysin or FLAGlysin102–198, and protein binding was assessed by ELISA. Symbols indicate the means ( SD) oftriplicate results in a representative experiment. (B) Binding of FLAGlysin or FLAGlysin102–198 to fibrinogen (far-Western blotting). Fibrinogen wasseparated by SDS-PAGE and stained with Coomassie blue (lanes 1 and 3) or transferred onto nitrocellulose and probed with FLAGlysin (lane 2)or FLAGlysin102–198 (lane 4). The three bands detected in lanes 1 and 3 correspond to the A�, B�, and � chains of fibrinogen. Binding of FLAGlysinor FLAGlysin102–198 to A� and B� chains was readily observed, with little or no binding to the � chain. (C) Inhibition of fibrinogen binding toimmobilized lysin by FLAGlysin or FLAGlysin102–198. The binding of fibrinogen (1 �g/ml) to immobilized lysin (5 �g/ml) was tested with buffercontaining 0 to 30 �M lysin or lysin102–198. Bound fibrinogen was detected by ELISA. *, P � 0.05, compared with 0 �M. Bars indicate the means( SD) of triplicate results in a representative experiment.

FIG. 4. Characterization of fibrinogen binding sites for lysin102–198. (A) Immobilized glycosylated fibrinogen or deglycosylated fibrinogen(deFg) (5 �g/ml) was incubated with the indicated concentrations of FLAGlysin102–198, and bound proteins were assessed by ELISA. (B) Bindingof FLAGlysin102–198 to recombinant fibrinogen chains. Each fibrinogen chain (expressed as a MalE fusion protein) was probed by far-Westernblotting for binding with FLAGlysin102–198. (C) Binding of recombinant fibrinogen chains to immobilized lysin102–198, as measured by ELISA.Symbols indicate the means ( SD) of triplicate results in a representative experiment.



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previously (Fig. 5A) (31). When wells containing immobilizedfibrinogen were preincubated with purified FLAGlysin102–198, theamount of SF100 binding to the wells was subsequently reduced.The level of inhibition was concentration dependent, with 4 �MFLAGlysin102–198 being sufficient to reduce SF100 binding to levelscomparable to those seen with PS1006.

We then examined whether purified lysin102–198 could inhibitbacterial binding to human platelets. SF100 demonstrated highlevels of binding to immobilized platelets, as described above.The preincubation of the platelet monolayers with purifiedFLAGlysin102–198 significantly reduced bacterial binding to im-mobilized platelets, with the level of inhibition being propor-tional to the amount of protein added (Fig. 5B). In controlstudies, FLAGlysin102–198 had no effect on the low levels offibrinogen or platelet binding seen with PS1006 (data not

shown). These data further indicate that SF100 binding tofibrinogen on platelet membranes is mediated by aa 102 to 198of lysinSM1.

Role of lysin in platelet aggregation. To assess further theeffect of lysin on the interaction of bacteria with platelets, wetested SF100 and PS1006 for the ability to induce plateletaggregation (Fig. 6). Both strains produced comparable levelsof aggregation in vitro, as measured by the maximum change inlight transmission (Fig. 6A). However, the time to the onset ofaggregation 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 plateletswith FLAGlysin102–198 affected subsequent aggregation bySF100 (Fig. 6B and C). We found that FLAGlysin102–198 inhib-ited platelet aggregation by SF100 in a concentration-depen-

FIG. 5. Inhibition of S. mitis binding to fibrinogen and platelets by lysin102–198. A total of 106 CFU of S. mitis strain SF100 was incubated withimmobilized human fibrinogen (A) or platelets (B) in the presence of FLAGlysin102–198 at the indicated concentrations (�M). Values representpercentages of SF100 binding and are the means of triplicate results from a representative experiment. Strain PS1006 (SF100lys) served as acontrol for low binding.

FIG. 6. Effect of lysinSM1 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 werepreincubated with the indicated concentrations of lysin102–198 followed by the addition of SF100. Representative data from a single donor areshown. (C) Effect of lysin on the time to the onset of platelet aggregation (lag time). The table shows results using platelets obtained from fourdifferent donors.



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dent manner, with as little as 4 nM polypeptide resulting in asignificant prolongation of the lag time. At 4 �M, FLAGlysin102–

198 completely blocked the aggregation of platelets from allfour donors tested. In control studies, lysin102–198 had no effecton aggregation by PS1006 (data not shown).

The above-described studies indicated that the binding oflysin to fibrinogen contributes to the induction of platelet ag-gregation by SF100. However, platelet aggregation by mostbacteria, including SF100, requires IgG specific for the organ-ism, which, after binding to the bacterial surface, triggers ac-tivation through the platelet Fc�RII receptor (30, 51–53; datanot shown). It was conceivable, therefore, that the prolongedlag times described above were due to the adsorption of anti-lysin antibodies by soluble FLAGlysin102–198, thereby reducingthe amount of IgG available for bacterial binding and Fc�RIIsignaling. To assess this possibility, we tested sera from severalhuman donors for the presence of antilysinSM1 IgG (Fig. 7).When examined by ELISA, high levels of antilysinSM1

IgG were present in all donor sera (n � 4). Antibodies tolysin102–198 were also detected but at considerably lower levels(Fig. 7A). In addition, the binding of IgG to immobilizedlysinSM1 was inhibited by preincubating the sera with purifiedlysinSM1 but not with lysin102–198 (Fig. 7B). These findings indi-cate that the inhibition of platelet aggregation by lysin102–198 isunlikely to be due to the adsorption of lysin-specific IgG andreduced Fc�RII-mediated activation. Rather, the prolongedlag times seen with the pretreatment of platelets withlysin102–198 is more likely to due to the inhibition of strep-tococcal binding to fibrinogen on the platelet surface, re-sulting in a delay in the onset of aggregation.

DISCUSSION

The pathogenesis of endocarditis is a multistep process com-prised of numerous host-pathogen interactions (14). Infectionof the endocardium is typically initiated by the attachment ofblood-borne organisms to damaged or prosthetic cardiac valvesurfaces that are covered by platelets and plasma proteins (34,59). This binding event is rapidly followed by the subsequentfurther deposition of platelets and other proteins, which, inconjunction with microbial proliferation, results in the forma-tion of macroscopic vegetations.

Our previous studies have identified several surface proteins

of S. mitis mediating binding to human platelets that are en-coded by a lysogenic bacteriophage, SM1 (30, 47). These pro-teins (PblA, PblB, and lysinSM1) are expressed during thephage lytic cycle and attach to the bacterial cell surface via theinteraction of their choline binding domains with lipoteichoicacid. The expression of these proteins by S. mitis is associatedwith increased virulence in animal models of endocarditis. Ofnote, recent studies indicated that homologs of PblA, PblB,and lysin are common among a range of Gram-positive patho-gens and that SM1 is a ubiquitous member of the oral micro-biota (3, 42, 60, 62). Since oral streptococci are among themost frequent causes of infective endocarditis, it is likely thatthese phage-encoded adhesins are highly conserved virulencefactors.

We recently found that lysinSM1 binds platelets in vitrothrough its interaction with fibrinogen on the platelet surface(47). However, it was unknown which region of the phageprotein mediated this interaction, and because lysinSM1 lackshomology with other known fibrinogen binding proteins, nocandidate regions were apparent. We now report that the re-gion of lysinSM1 corresponding to residues 102 to 198 mediatesthis interaction and that lysin102–198 has fibrinogen bindingproperties that are comparable to those of the full-length pro-tein. In particular, the two proteins appeared to have compa-rable affinities for fibrinogen. Both full-length lysin andlysin102–198 selectively bound the A� and B� chains of fibrin-ogen, and the binding of lysinSM1 was effectively blocked by thepolypeptide. These results indicate that the fibrinogen bindingproperties 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 asthe ClfA, FnbpA, SdrG, and Aaa proteins of Staphylococcusand the M proteins of Streptococcus pyogenes (12, 13, 15, 20, 21,57). Among the best characterized of the staphylococcal fibrin-ogen binding proteins are ClfA and SdrG, where the solutionof their crystal structures in complex with fibrinogen-derivedpeptides has revealed the structural basis for binding. BothClfA and SdrG contain two Ig-like domains that form a pocketfor fibrinogen binding via a “dock, lock, and latch” mechanism(38). These proteins differ, however, in that ClfA recognizesthe C terminus of the � chain, whereas SdrG recognizes the Nterminus of the � chain (17, 38). The crystal structure of the

FIG. 7. Levels of antilysin IgG in human sera and inhibition of antibody binding to immobilized lysinSM1 by soluble lysinSM1 or lysin102–198.(A) Immobilized lysinSM1 or lysin102–198 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 (�M) of lysinSM1 or lysin102–198 and then tested individually for antibodybinding to immobilized lysinSM1 by ELISA.



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M1 protein of S. pyogenes in complex with fibrinogen fragmentD was reported recently (28). These proteins were found toform a cross-like complex comprised of a coiled-coil dimer ofM1 bound to four molecules of fragment D. Less is knownabout the details of fibrinogen binding by other bacterial pro-teins, due to the lack of structural information. Proteins suchas Eap and Efb have been shown to bind the A� chain througha series of tandem repeats, but how these subdomains form acomplex with fibrinogen has not been determined (12, 18, 55).

Although the precise structural requirements for fibrinogenbinding by lysinSM1 are not fully known, the predicted aminoacid sequence of lysin102–198 has no significant homology toany of the above-described fibrinogen binding proteins.In addition, structure prediction searches (using PSI-BLAST,PHYRE, and HHPRED) (1, 23, 50) failed to identify anypotential structural similarities between lysinSM1 and thesefibrinogen binding proteins (data not shown), further indicat-ing that lysinSM1 binds fibrinogen by a distinct mechanism. Asfor other bacterial proteins known to bind fibrinogen, onepossible homolog of lysinSM1 is choline binding protein E(CbpE) of S. pneumoniae. CbpE is a cell wall-associated pro-tein and a member of the metallo-�-lactamase superfamily thathas been shown to bind fibrinogen in vitro (16). Although thestructure of CbpE has been solved, the regions of this proteinsinvolved in fibrinogen binding have not been identified. CbpEshares only 20% overall amino acid identity with lysinSM1, andan alignment of the proteins indicates that the region of CbpEcorresponding to lysin102–198 has even less identity (17%).However, secondary-structure analysis predicts that these pro-teins share a high level of similarity in folding, suggesting thatthey could bind fibrinogen by a similar mechanism.

In addition to mediating platelet binding, we found thatphage lysin contributes to platelet aggregation by S. mitis. Theloss of lysin expression was associated with a prolonged lagtime, which also reflects the time to the onset of platelet acti-vation (15, 27, 29, 34, 36). The preincubation of platelets withlysin102–198 prior to mixing with whole bacteria also delayedaggregation, further indicating that fibrinogen binding by lysincontributes to aggregation. These findings are consistent withprevious studies of several staphylococcal proteins (e.g., ClfA,ClfB, and FnBpA), which were also found to promote plateletaggregation 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 tothe bacterial surface, interacts with the platelet Fc�RII toinduce activation and aggregation (14, 15, 27). This findingraised the possibility that the effect of lysin on the lag time mayhave been due to its interaction with specific antibodies and theengagement of Fc�RII, rather than fibrinogen binding. How-ever, although sera from our donors did contain antibodies tofull-length lysin, only a minor component appeared to be di-rected against the fibrinogen binding domain. It is thus unlikelythat antibodies specific for this domain contribute significantlyto activation. In contrast, since even nanomolar concentrationsof lysin102–198 in solution prolonged the lag time, it is likely thatthe lysin on the bacterial surface accelerates platelet aggrega-tion through its interaction with fibrinogen.

In summary, the lysin of bacteriophage SM1 appears to be anovel fibrinogen binding protein. The binding region oflysinSM1 has no predicted structural similarities to previously

characterized binding domains of other fibrinogen binding pro-teins. Thus, the precise basis for lysin binding and the promo-tion of aggregation is unknown and will require additionalstructural analyses, which are now in progress.

ACKNOWLEDGMENTS

This study was supported by the VA Merit Review program; bygrants R01-AI41513 and R01-AI057433 (P.M.S.) from the NationalInstitutes of Health (which were administered by the NorthernCalifornia Institute for Research and Education); by a fellowshipaward from the American Heart Association, Western Affiliate(H.S.S.); and by resources of the Veterans Affairs Medical Center,San Francisco, CA.

We thank Susan Lord for providing us with plasmids for expressingfibrinogen.

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