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. 2005 Oct;73(10):6629-38.
doi: 10.1128/IAI.73.10.6629-6638.2005.

Functions of cell surface-anchored antigen I/II family and Hsa polypeptides in interactions of Streptococcus gordonii with host receptors

Nicholas S Jakubovics  1 , Steven W Kerrigan, Angela H Nobbs, Nicklas Strömberg, Craig J van Dolleweerd, Dermot M Cox, Charles G Kelly, Howard F Jenkinson
Affiliations

Functions of cell surface-anchored antigen I/II family and Hsa polypeptides in interactions of Streptococcus gordonii with host receptors

Nicholas S Jakubovics et al. Infect Immun. 2005 Oct.

Abstract

Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surface receptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements into biologically productive phenomena.

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Figures

FIG. 1.
FIG. 1.
(A) Organization of the hsa locus in S. gordonii Challis (strain DL1) and insertion site of aphA3 for generating strain UB1545 Δhsa. The arrangement of genes (derived from the genomic sequence of S. gordonii CH1 at www.tigr.org) potentially involved in Hsa posttranslational processing (open arrows) and secretion (shaded arrows) is shown (also see references and 54). The glycosyltransferase (gly) gene contains a frameshift mutation (asterisk) predicted to result in production of a truncated polypeptide. A stem-loop (rho-independent transcriptional terminator) and a putative promoter element (P) are present between the hsa and gly genes. (B) Transcription of gly in strains DL1 and UB1545 was monitored by quantitative RT-PCR. No product was observed in the absence of reverse transcriptase (RT). Measurements of thiol peroxidase (tpx) and ScaR metallorepressor protein (scaR) gene transcript levels were included as controls.
FIG. 2.
FIG. 2.
Expression of Hsa and AgI/II polypeptides by S. gordonii DL1, UB1545 Δhsa, and UB1360 Δ(sspA sspB). Cell wall protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted onto a nitrocellulose membrane. (A) sWGA detection of Hsa, indicated by an arrow. (B) SspA and SspB (AgI/II family) proteins visualized with polyclonal antibodies to AgI/II, indicated by an arrow (the two lower-molecular-mass bands are fragments). (C) Twofold dilutions of intact cells dot blotted onto nitrocellulose and reacted with antibodies to surface proteins (SLS extract) of S. gordonii DL1, with sWGA, or with polyclonal antibodies to AgI/II.
FIG. 3.
FIG. 3.
Transcription of the hsa gene in S. gordonii DL1 and strain UB1360 Δ(sspA sspB) assessed by quantitative RT-PCR. Amplification products from tpx and scaR mRNAs were included as controls. Bands were subjected to densitometric analyses, and hsa mRNA product levels were calculated relative to the tpx and scaR mRNA product levels in the corresponding strain. Experiments were repeated three times, and a representative gel from one experiment is shown.
FIG. 4.
FIG. 4.
Adhesion of S. gordonii strains to immobilized gp340. Binding of DL1 (⧫), UB1360 Δ(sspA sspB) (▪), UB1545 Δhsa (▴), or UB1552 Δhsa Δ(sspA sspB) (○) to gp340 immobilized onto plastic wells was determined following crystal violet staining of bound cells (see Materials and Methods). Experiments were repeated at least three times, and the results of one representative experiment are shown; the data points are means for triplicate samples, and the standard deviations were within ±10%.
FIG. 5.
FIG. 5.
Modulation of streptococcal adhesion to gp340 by sialidase. (A) S. gordonii DL1 and UB1360 Δ(sspA sspB) mutant; (B) S. mutans NG8 and 834 ΔspaP; (C) L. lactis MG1363 wild type or strains expressing AgI/II polypeptide SspA, SspB, or PAc. Bacterial adhesion to 0.2 μg immobilized gp340, pretreated with buffer (control) or sialidase, was quantified by crystal violet staining of bound cells. Means and standard deviations of three independent experiments are shown.
FIG. 6.
FIG. 6.
Aggregation of S. gordonii by fluid-phase gp340. Purified gp340 (1.2 μg) was added to bacterial cell suspensions (1 × 109 cells/ml), and the extent of aggregation (reduction in OD600) at 37°C was measured at intervals. Symbols: ⧫, DL1; ▴, UB1360; ▪, UB1545; ○, UB1552; ⋄, DL1 control (no gp340). Samples were run in triplicate in three experiments, and the results from one representative experiment are shown.
FIG. 7.
FIG. 7.
Adhesion of S. gordonii DL1 (wild type), UB1360 Δ(sspA sspB), UB1545 Δhsa, and UB1552 Δhsa Δ(sspA sspB) to HEp-2 epithelial cells. The percentages of streptococcal cells (input, 105 cells/ml) that were bound after 2 h of incubation at 37°C were determined by viable counting. The values are the means and standard deviations of three independent experiments.
FIG. 8.
FIG. 8.
Platelet adhesion by S. gordonii DL1 (wild type), UB1360 Δ(sspA sspB), UB1545 Δhsa, and UB1552 Δhsa Δ(sspA sspB). Equivalent numbers of streptococcal cells (3 × 108 cells) were immobilized onto microtiter plate wells, and the adhesion of purified human platelets (2 × 108 cells/ml) was measured by an enzymatic assay as described in Materials and Methods. The data are the means and standard deviations of three independent experiments.
FIG. 9.
FIG. 9.
S. gordonii-mediated platelet aggregation. S. gordonii cells (7 × 109 cells/ml) were mixed with human platelets (2 × 108 cells/ml) in plasma, and aggregation was expressed as a percentage of the control sample aggregation induced with arachidonic acid. Means and standard deviations are shown, based on five independent assays. No aggregation occurred with strain UB1552 Δhsa Δ(sspA sspB).
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