Streptococcus mitis Phage-Encoded Adhesins Mediate Attachment to α2-8-Linked Sialic Acid Residues on Platelet Membrane Gangliosides

Bacterial strains and culture conditions.The bacterial strains used in this study are S. mitis SF100 and its isogenic ΔpblA ΔpblB variant strain, S. mitis PS344 (3). Of note, SF100 and PS344 have no detectable sialidase activity (unpublished data), and both strains grow equally well in broth culture (3). Bacteria were grown statically at 37°C in Todd-Hewitt broth (Difco Laboratories) or on sheep blood agar (Hardy) at 37°C in a 5% CO₂ environment.

Quantitative assay for binding to immobilized platelets.Overnight cultures were diluted 1:10 in fresh Todd-Hewitt broth, incubated for 1 h at 37°C, and then exposed to UV light (λ = 312 nm) for 3 min. The cultures were incubated at 37°C for an additional 4 h, followed by the harvesting of bacteria by centrifugation at 8,200 × g for 15 min and washing three times in phosphate-buffered saline (PBS).

The binding of streptococci to human platelets was assessed quantitatively as described previously (20). In brief, washed, fixed human platelets were immobilized in poly-l-lysine-coated, 22-mm-diameter tissue culture wells, producing monolayers of 75 to 90% confluence. To reduce nonspecific adherence, the wells were treated with a casein solution (blocking reagent; Roche) in PBS for 2 h at room temperature. After the blocking solution was removed by aspiration, the wells were inoculated with approximately 1 × 10⁷ CFU of streptococci suspended in 1 ml of PBS and incubated at 37°C for 2 h, with gentle rocking to enhance mixing. The viability of bacterial cells in PBS was tested at 30-min intervals over the 2-h incubation period and was found not to change over time, and there was no difference in survival between the wild-type SF100 cells and PS344 cells. Unbound bacteria were then removed by washing with PBS, and bound bacteria were recovered by scraping and resuspension in 1 ml PBS. The number of organisms bound was determined by plating serial dilutions of the suspension onto blood agar, and binding was expressed as a percentage of the inoculum. All studies were carried out in triplicate on three separate occasions, using platelets from a different human donor on each day. Differences in platelet binding were compared by the unpaired t test.

Quantitative assay for binding to deglycosylated platelets.Platelets monolayers were washed three times in PBS and then incubated with a final concentration of 5 mU/ml of N-glycanase, O-glycanase, sialidase A, sialidase C, or sialidase V (Glyko) for 16 h at 37°C. Platelets were washed a further three times with PBS, and all wells were incubated with a solution of 2% formaldehyde in PBS for 1 h at room temperature to inactivate any remaining enzyme. Control wells were also treated with 2% formaldehyde in PBS. The platelet monolayers were then washed three times with PBS and the binding of bacterial cells to platelets was assayed as described above.

Inhibition of binding to platelets by antibodies.Platelet monolayers were washed three times in PBS and then incubated with a final concentration of 0.5 μg/ml anti-GD3 monoclonal antibody (MAb) 4F6 (immunoglobulin G3 [IgG3]), an isotype control IgG3 antibody MG3-35 or an anti-GP1b IgG1 MAb HIP1 (Abcam) for 1 h at room temperature with gentle agitation. Platelets were subsequently washed three times in PBS, and the binding of bacterial cells to treated platelets was assayed as described above.

Concentration-dependent inhibition of binding to platelets by antibodies.Platelet monolayers were washed three times in PBS and then incubated with 10-fold dilutions of 5 μg/ml of MAb 4F6 starting at a 10⁻² dilution (0.5 μg/ml) for 1 h at room temperature with gentle agitation. Platelets were washed in PBS, and bacterial binding was assessed as described above.

Inhibition of binding to platelets by gangliosides.Overnight cultures of S. mitis were processed as described above and then incubated with a final concentration of 0.35 mM ganglioside (EMD Biosciences) for 1 h at room temperature with gentle agitation. The bacteria were then washed three times in PBS, and the binding of treated bacterial cells to platelets was assayed as described above. The gangliosides used and their structures are listed in Table 1.

Concentration-dependent inhibition of binding to platelets by GD3.Overnight cultures of S. mitis were processed as described above and then incubated with serial 10-fold dilutions of 3.5 mM GD3 (10⁰ to 10⁻⁴ dilution) for 1 h at room temperature. The binding of bacteria to platelets was then assessed as detailed above.

Direct binding of bacteria to immobilized gangliosides.Thirty-five micromoles of each ganglioside was added to a flat well in a 96-well microtiter dish and dried for 16 h at 37°C. The wells were then blocked with 200 μl of casein solution for 2 h at room temperature.

Bacteria were grown as described above, harvested, washed in PBS, and suspended in PBS to 1 × 10⁷ CFU/ml. Fifty microliters of each bacterial suspension was added to each well and allowed to adhere for 1 h at room temperature with gentle agitation. Plates were subsequently washed three times with 200 μl PBS. Adherent bacteria were detached from the plate by incubation of the wells with trypsin for 10 min at room temperature, and the trypsin reaction was stopped by the addition of 50 μl fetal bovine serum to each well. The number of organisms bound was determined by plating serial dilutions of the bacterial suspension onto blood agar, and binding was expressed as a percentage of the inoculum. All studies were carried out in triplicate. Differences in binding were compared by the unpaired t test.

Binding of PlbA and PbB to immobilized gangliosides.Asialo GM1 and GD3 affinity binding studies were performed as described previously (12). In brief, 2 mM of GD3 or asialo GM1 was bound separately to 200 μl of prewashed octyl-Sepharose CL 4B (GE Healthcare) in a solution of methanol-water (1:1, vol/vol) and 0.1 M KCl for 4 h at room temperature. The Sepharose beads were washed extensively with PBS and then incubated for 1 h with clarified supernatants of SF100 or PS344 cultures, with rotation at room temperature. The beads were then washed repeatedly with PBS to remove unbound material until no protein could be detected in the recovered fluid, as measured by UV light absorbance. Octyl-Sepharose CL 4B served as a control for nonspecific adsorption. Bound proteins were eluted with 6 M urea.

All samples were added to lithium dodecyl sulfate sample buffer (Invitrogen) and separated on 3 to 8% Tris-acetate polyacrylamide gels (Invitrogen). The separated proteins were transferred to nitrocellulose membranes (Invitrogen), blocked with 1× blocking reagent (Roche), and probed with anti-PblA antibodies or anti-PblB antibodies. Bound antibodies were detected with anti-goat IgG horseradish peroxidase (Sigma) and SuperSignal chemiluminescent substrate (Pierce).

Desialylation of platelets results in a reduced platelet binding by S. mitis.In pilot studies examining the platelet receptor for S. mitis SF100, several proteins known to be receptors for other viridans group streptococci and gram-positive pathogens were tested for bacterial binding. S. mitis SF100 did not bind to fibrinogen, fibronectin, collagen, or the platelet glycoprotein GPIb or GPIIb/IIIa (data not shown). In addition to these protein targets, we also examined the contribution of surface-exposed carbohydrates to platelet binding by SF100.

We first assessed the impact of treating platelet monolayers with sialidase A or N-glycanase and O-glycanase on platelet binding by S. mitis SF100 and its isogenic ΔpblA ΔpblB variant strain in order to determine whether S. mitis adheres to glycosylated residues on the surface of platelets. Sialidase A is a recombinant sialidase from Arthrobacter ureafaciens that specifically cleaves nonreducing terminal branched and unbranched sialic acids from glycoproteins and glycolipids. N-Glycanase is a recombinant glycosidase from Elizabethkingia meningoseptica that cleaves asparagine-linked oligosaccharides from glycoproteins. O-Glycanase is a recombinant glycosidase from Streptococcus pneumoniae that releases unmodified oligosaccharides attached to a serine or threonine residue in glycoproteins.

As is shown in Fig. 1, desialylation of platelets significantly reduced the binding of S. mitis SF100 to platelets (P < 0.0001), whereas it had a negligible effect on the residual background level of binding by PS344. In contrast, pretreatment of the platelets with N- and O-glycanases had no impact on SF100 or PS344 binding to platelets. These results indicate that the primary host receptor for S. mitis SF100 is sialylated and that the phage proteins PblA and PblB mediate adherence of SF100 to sialic acid residues. The inability of N- and O-glycanase treatment to reduce S. mitis platelet binding also indicates that the sialylated glycans to which S. mitis is binding are more likely to form part of a glycolipid rather than a glycoprotein, although it is possible that more-complex O-linked carbohydrates may partially contribute to the interaction of S. mitis SF100 with platelets.

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FIG. 1.

Desialylation of human platelets reduces binding by S. mitis. Binding of SF100 (□) to untreated platelets (lane 1), platelets pretreated with sialidase A (lane 2), platelets treated with N- and O-glycanase (lane 3), and platelets treated with N- and O-glycanase and sialidase A (lane 4). Binding of PS344 (▪) to untreated platelets (lane 5), platelets pretreated with sialidase A (lane 6), platelets treated with N- and O-glycanase (lane 7), and platelets treated with N- and O-glycanase and sialidase A (lane 8). Values are expressed as a percentage of wild-type binding to untreated platelets (mean ± SD).

Enzymatic removal of α2-8-linked sialic acid residues from platelets reduces S. mitis platelet binding.To further characterize the type of sialic acid linkages bound by S. mitis SF100, platelets were treated with sialidases with differing linkage specificities. Sialidase V is a sialidase from Vibrio cholerae which cleaves α2-3-, α2-6-, and α2-8-linked sialic acids from oligosaccharides. Sialidase C is a recombinant sialidase from Clostridium perfringens which cleaves α2-3- and α2-6-linked sialic acids. Pretreatment of platelet monolayers with sialidase A or sialidase V resulted in a significant reduction in SF100 binding to platelets (P = 0.0241 or 0.0018, respectively) (Fig. 2). In contrast, treatment of platelet monolayers with sialidase C had no significant effect on platelet binding by SF100. In control studies, none of the sialidases tested altered the already low levels of binding by PS344. These results indicate that α2-8-linked sialic acid constitutes the main platelet receptor for SF100.

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FIG. 2.

Cleavage of α2-8-linked sialic acid from platelet surfaces results in a reduction of S. mitis binding. Binding of SF100 (□) to untreated human platelets (lane 1) or platelets pretreated with sialidase A (lane 2), sialidase V (lane 3), or sialidase C (lane 4). Binding of PS344 (▪) to platelets (lane 5) or platelets pretreated with sialidase A (lane 6), sialidase V (lane 7), or sialidase C (lane 8). Values presented are expressed as a percentage of wild-type binding to untreated platelets (mean ± SD). Data represent three experiments performed in triplicate on different occasions from a different donor each time. Note that these donors are different from those shown in Fig. 1.

Antibody to ganglioside GD3 blocks S. mitis SF100 platelet binding.Ganglioside GD3 is the only reported molecule containing α2-8-linked sialic acid on the platelet membrane surface. To elucidate whether this was the receptor for S. mitis SF100, platelets were preincubated with an IgG3 MAb to GD3. The ability of SF100 cells to adhere to the antibody-treated platelets was then tested. As shown in Fig. 3A, the MAb 4F6 reduced the adherence of SF100 to platelets by 45% (P = 0.0052). In contrast, this antibody did not affect the residual binding of S. mitis strain PS344. Additionally, treatment of platelets with a MAb to the glycosylated platelet membrane protein GPIb (IgG1 MAb HIP1) as a control for nonspecific binding of SF100 to sialylated proteins did not block binding of strain SF100 to platelets. Treatment of platelets with an isotype control IgG3 MAb did not affect S. mitis binding these cells (data not shown). Additionally, the ability of anti-GD3 MAb 4F6 to block the interaction of S. mitis strain SF100 to platelets was concentration dependent and saturable (Fig. 3B). These data confirm that the ganglioside GD3 is a receptor for S. mitis platelet binding and that the phage proteins PblA and PblB mediate binding to this receptor.

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FIG. 3.

Antibodies to ganglioside GD3 block S. mitis binding to platelets. (A) Binding of SF100 (□) to untreated platelets (lane 1), platelets pretreated with a murine MAb to ganglioside GD3 (lane 2), or a murine MAb to platelet glycoprotein GPIb (lane 3) and binding of PS344 (▪) to untreated platelets (lane 4) platelets pretreated with a murine MAb to ganglioside GD3 (lane 5) or a murine MAb to platelet glycoprotein GPIb (lane 6). (B) Concentration-dependent inhibition of S. mitis (□) and PS344 (▪) binding to platelets by a murine MAb to GD3. Values presented are expressed as a percentage of wild-type binding to untreated platelets (mean ± SD).

Preincubation of S. mitis SF100 with ganglioside GD3 blocks platelet binding.To more fully assess the role of gangliosides in platelet binding, S. mitis SF100 and PS344 were incubated with a variety of differentially glycosylated gangliosides (Table 1) and subsequently tested for their abilities to adhere to platelets. Pretreatment of SF100 with asialo GM1 or GM1 did not significantly affect the binding of this strain to platelets (Fig. 4). However, treatment of SF100 with GM3 reduced binding by 35%. Moreover, treatment of SF100 with GD3, which is the ganglioside in platelet membranes containing α2-8-linked sialic acid, caused a 45% reduction in platelet binding (P = 0.0031). GD3 also blocked the interaction of S. mitis SF100 with platelets in a concentration-dependent manner, as shown in Fig. 4B.

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FIG. 4.

Effect of gangliosides on platelet binding by S. mitis. (A) Strains SF100 (□) and PS344 (▒) were incubated with buffer or gangliosides and then tested for binding to platelets. Gangliosides tested were as follows: asialo GM1, GM1, GM3, GD3, GD1a, GD1b, GT1b, and GQ1b. (B) Strains SF100 (□) and PS344 (▪) were incubated with 10-fold dilutions of ganglioside GD3 and then tested for binding to platelets. Values are expressed as a percentage of wild-type binding to platelets (mean ± SD).

Further analysis of the ability of different gangliosides to block SF100 platelet binding revealed that preincubation of SF100 with gangliosides containing α2-8-linked sialic acids (Fig. 4) blocked binding to platelets. Like GD3, ganglioside GD1a (Fig. 4) is also disialylated, but both sialic acids are α2-3 linked. Preincubation of SF100 with GD1a did not result in a significant inhibition of platelet binding. In parallel studies, none of the gangliosides tested affected the residual binding of PS344 to human platelets. These data further indicate that binding of SF100 to platelets is specific for the α2-8-linked sialic acids in ganglioside GD3 and that this interaction is mediated by PblA and PblB.

Direct binding of S. mitis SF100 to ganglioside GD3 requires PblA/PblB.To assess the direct interaction of strains SF100 and PS344 with gangliosides, we compared the adherences of these strains to gangliosides immobilized on microtiter dishes. As shown in Fig. 5A, there is no significant difference in the relative adherence of either strain to lactosylceramide, a structural precursor to GM3 and GD3 that does not contain sialic acid. Similarly, no significant difference was seen in the binding of SF100 versus PS344 to ganglioside GM3 (Fig. 5B). However, the binding of strain PS344 to the ganglioside GD3 was reduced by 70% compared to that of the wild-type strain SF100 (P = 0.0223) (Fig. 5C). These data show that SF100 binds directly to ganglioside GD3 and that this binding is mediated by the phage proteins PblA and PblB.

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FIG. 5.

Binding of S. mitis to immobilized gangliosides. Strains SF100 (□) and PS344 (▒) were assayed for binding to lactosyl ceramide (A), GM3 (B), and GD3 (C). Values are expressed as a percentage of wild-type binding to ganglioside (mean ± SD).

PblA and PblB proteins bind directly to ganglioside GD3.In order to verify that PblA and PblB proteins bind directly to GD3, clarified cell culture supernatants of S. mitis SF100 were mixed with either GD3 or asialo GM1 immobilized on octyl-Sepharose CL-4B. The Sepharose was then extensively washed, and then any proteins bound to GD3 or asialo GM1 were recovered by treatment with urea. As is shown Fig. 6, both PblA and PblB were affinity purified with GD3 Sepharose. In contrast, neither PblA nor PblB bound to asialo GM1 immobilized on Sepharose (Fig. 6B) or to octyl-Sepharose CL-4B alone (data not shown). These findings indicate that PblA and PblB proteins interact directly with GD3 and that this interaction is specific.

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FIG. 6.

Direct binding of PblA and PblB proteins to GD3. Clarified culture supernatants of SF100 were passed over GD3 Sepharose (A) or asialo GM1 Sepharose (B) in order to assess the direct binding of PblA and PblB. Lane 1, supernatant; lane 2, flowthrough; lane 3, first column wash; lane 4, final column wash; lane 5, eluate. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes and probed with antibodies to PblA (αPblA) or PblB (αPblB).