Journal List > J Bacteriol Virol > v.44(4) > 1034148

Jang, Lin, Lim, Kim, and Seo: Binding of the Streptococcus gordonii Surface Glycoprotein Hsa to α(2–3) Linked Sialic Acid Residues on Fibronectin

Abstract

The binding of microorganisms to platelets is a critical step in the development of infective endocarditis. In Streptococcus gordonii, this binding is mediated in part by serine-rich repeat proteins, which interact directly with sialic acid residues located on GPIIb receptors in the platelet membrane. In this study, we found that S. gordonii DL1 strain binds to platelets through bridging between sialic acid residue of fibronectin and serine-rich repeat protein (Hsa). Pretreatment of fibronectin with sialidases specific for α(2–3)-linked sialic acids was shown to significantly inhibit binding of the DL1 strain and the binding region(BR) of Hsa protein. Similarly, pre-incubation of bacteria or BR of Hsa with α(2–3)-sialyl-N-acetyllactosamine blocked fibronectin binding in the DL1 strain, but not the M99 strain. Together, these data show that the α(2–3)-sialic acid residues of fibronectin play an important role in the binding of S. gordonii DL1 to fibronectin through interactions with the Hsa receptor. This interaction is thought to play an important role in the development of pathogenic endocarditis, and may represent an important therapeutic target for the treatment of infective endocarditis.

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Figure 1.
Schematic drawing of the Srr proteins GspB and Hsa of S. gordonii. *S: signal sequence; SRR1 and SRR2: SRR regions; BR: binding region; LPxTG: cell wall anchoring motif.
jbv-44-317f1.tif
Figure 2.
Binding of S. gordonii DL1 to fibronectin. (A) Binding of S. gordonii DL1 to immobilized plasminogen, thrombin, laminin, collagen, fibrinogen, and fibronectin (1 μM/well). (B) Binding of S. gordonii, S. sanguinis, and Group B streptococci to immobilized fibronectin. All values are expressed as a percentage of S. gordonii DL1 binding to platelets (mean ± SD).
jbv-44-317f2.tif
Figure 3.
S. gordonii DL1 binding to fibronectin is mediated by the interaction of the Srr protein Hsa to sialic acid residues of fibronectin. (A) Streptococcus gordonii strains M99, DL1, and 70-40 were compared with their respective srr mutants for fibronectin binding (1 μM). (B) Binding of S. gordonii and its isogenic mutant strain (Δsrr) to fibronectin treated (+) or untreated (−) with sialidase A (5 mU/well). All values are expressed as a percentage of wild-type binding to untreated fibronectin (means ± SD).
jbv-44-317f3.tif
Figure 4.
Binding of Hsa and GspB-BRs to the α(2–3)-linked sialic acid residues of fibronectin. (A) Binding of purified GST-Hsa-BR and GST-GspB-BR to immobilized fibronectin (1 μM). Bound proteins were detected with anti-GST antibodies. (B) Binding of purified GST-Hsa-BR and GST-GspB-BR to fibronectin treated (+) or untreated (−) with sialidase A, V, I, or S (5 mU/well). Bars indicate the means ± SD.
jbv-44-317f4.tif
Figure 5.
Effect of α(2–3)-linked sialyl-N-acetyllactosamine on fibronectin binding by S. gordonii DL1 and GST-Hsa-BR. (A) S. gordonii strains M99 and DL1 were incubated with PBS, α(2–3)-sialyl-N-acetyllactosamine (10 μM), or α(2–6)-sialyl-N-acetyllactosamine (10 μM) and then tested for binding to immobilized fibronectin. All values are expressed as a percentage of wild-type binding to platelets (means ± SD). (B) GST-GspB-BR and GST-Hsa-BR were incubated with PBS, α(2–3)-sialyl-N-acetyllactosamine (10 μM), or α(2–6)-sialyl-N-acetyllactosamine (10 μM) and then tested for binding to immobilized fibronectin. Bars indicate the means ± SD.
jbv-44-317f5.tif
Table 1.
Bacteria strains and plasmids
Strain or plasmid Genotype or description Source
Escherichia coli
 DH5 Frm+Ø80dlacZΔM15 Gibco BRL
 BL21 (DE3) Expression host, inducible T7 RNA polymerase Novagen
Streptococcus gordonii
 DL1 Endocarditis clinical isolate [20]
 M99 Endocarditis clinical isolate [20]
 72-40 Endocarditis clinical isolate [20]
 G9B Endocarditis clinical isolate This study
 AMS12 Endocarditis clinical isolate This study
 DL1Δhsa Hsa isogenic deletion mutant [20]
 M99ΔgspB gspB isogenic deletion mutant [20]
Streptococcus sanguinis
 PS478 Endocarditis clinical isolate This study
 10556 Endocarditis clinical isolate This study
 804 Endocarditis clinical isolate This study
Streptococcus agalactiae
 COH31 Serotype III, clinical isolate [24]
Plasmids
 pGEX-hsa Vector for expression of Hsa [27]
 pGEX-gspB Vector for expression of GspB [27]
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