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. 2004 Jul;72(7):3876-82.
doi: 10.1128/IAI.72.7.3876-3882.2004.

Functional analysis of the Streptococcus gordonii DL1 sialic acid-binding adhesin and its essential role in bacterial binding to platelets

Yukihiro Takahashi  1 , Ayako Yajima, John O Cisar, Kiyoshi Konishi
Affiliations

Functional analysis of the Streptococcus gordonii DL1 sialic acid-binding adhesin and its essential role in bacterial binding to platelets

Yukihiro Takahashi et al. Infect Immun. 2004 Jul.

Abstract

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The sialic acid-binding adhesin of Streptococcus gordonii DL1 was previously associated with the hsa gene encoding a 203-kDa protein. The predicted protein sequence consists of an N-terminal nonrepetitive region (NR1), including a signal sequence, a relatively short serine-rich region (SR1), a second nonrepetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats, and a C-terminal cell wall anchoring domain. In the present study, the contributions of SR1, NR2, and SR2 to Hsa-mediated adhesion were assessed by genetic complementation. Adhesion of an hsa chromosomal deletion mutant to sialic acid-containing receptors was restored by plasmids containing hsa constructs encoding Hsa that lacked either the N- or C-terminal portion of SR2. In contrast, hsa constructs that lacked the coding sequences for SR1, NR2, or the entire SR2 region failed to restore adhesion. Surface expression of recombinant Hsa was not affected by removal of SR1, NR2, or a portion of SR2 but was greatly reduced by complete removal of SR2. Wheat germ agglutinin, a probe for Hsa-specific glycosylation, reacted with recombinant Hsa lacking SR1, NR2, or SR2 but not with recombinant Hsa lacking both SR1 and SR2. Significantly, the aggregation of human platelets by S. gordonii DL1, an interaction implicated in the pathogenesis of infective endocarditis, required the expression of hsa. Moreover, neuraminidase treatment of the platelets eliminated this interaction, further supporting the hypothesis that Hsa plays an essential role in the bacterium-platelet interaction.

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Figures

FIG. 1.
FIG. 1.
Alignment of the physical map of the 7.4-kb HindIII-SphI fragment of S. gordonii DL1 chromosomal DNA in pAS8741 with the nonrepetitive regions (NR1 and NR2), serine-rich repetitive regions (SR1 and SR2), and cell wall anchoring domain (CWAD) of the encoded Hsa protein. a.a, amino acid.
FIG. 2.
FIG. 2.
Restoration of Hsa-mediated adhesion by genetic complementation of S. gordonii CM100 with plasmids expressing hsa (i.e., pAS8741) or hsa deletion constructs. The positions of the nonrepetitive regions (NR1 and NR2), serine-rich repetitive regions (SR1 and SR2), and cell wall anchoring domain (CWAD) of Hsa are indicated above the diagram of the complete protein encoded by pAS8741. The V-shaped lines indicate the regions removed from recombinant proteins by in-frame deletions in plasmid-borne hsa. The hemagglutinating activity of each plasmid-bearing transformant is expressed relative to that of CM100(pAS8741). Bacterial binding to immobilized sialoglycoconjugates is indicated by a plus or minus sign.
FIG. 3.
FIG. 3.
Binding of anti-Hs (1:100), WGA-biotin (50 μg/ml), and anti-DL1 (1:1,000) to S. gordonii CM100 harboring different plasmids. Wild-type strain DL1 and the hsa insertional mutant strain EM230 were also included. Washed bacteria were spotted on three separate nitrocellulose membranes. Each membrane was incubated with primary antibody followed by peroxidase-conjugated goat anti-rabbit IgG or with WGA-biotin followed by avidin. The membrane labeled with anti-DL1 was included as a positive control for each strain.
FIG. 4.
FIG. 4.
Western and lectin blotting of streptococcal cell wall (CW) and protoplast (PP) proteins of S. gordonii DL1 and strain CM100 harboring different plasmids. The material in each sample was obtained from 1 ml of bacterial culture. Sonic extracts (Sonic) (50 μg of total protein/lane) of CM100(pAS8165), CM100(pAS8164), and CM100(pAS40S) were also examined. SDS-PAGE was performed with 5% polyacrylamide gels (upper panels) or 10% polyacrylamide gels (lower panels). Nitrocellulose transfers were incubated with anti-Hs (1:100) followed by peroxidase-conjugated goat anti-rabbit IgG or with WGA-biotin (50 μg/ml) followed by avidin. The boundary between the stacking and separating gels (asterisk) and the positions of molecular mass markers are indicated on the left.
FIG. 5.
FIG. 5.
Essential role of Hsa in adhesion of S. gordonii DL1 to human platelets. (A) Microtiter wells containing washed platelets and bacteria, showing visible aggregation in wells containing bacteria that surface express Hsa (wild-type strain DL1 and strain CM100 harboring pAS8741) and no aggregation in wells containing bacteria that do not produce Hsa (strain EM230, an hsa insertional mutant, and strain CM100 harboring control plasmid pAS40S). (B) Aggregation of untreated platelets in TBS-EDTA (U) (cross-hatched bars) or in TBS-Ca2+ (C) (open bars) and aggregation of neuraminidase-treated platelets (N) (solid bars).
FIG. 6.
FIG. 6.
Comparison of the accessory sec locus downstream of gspB in S. gordonii M99 (1) with a similar locus downstream of hsa in S. gordonii Challis (i.e., strain DL1). Nucleotide sequence identities between corresponding genes were calculated with the program BESTFIT (GCG Wisconsin package). The deletion mutant strain CM100, which was used in genetic complementation studies, lacked the 9.2-kb HindIII fragment identified in the open reading frame diagram of strain Challis.
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