Streptococcus pyogenes antigen I/II-family polypeptide AspA shows differential ligand-binding properties and mediates biofilm formation

Abstract

The streptococcal antigen I/II (AgI/II)-family polypeptides are cell wall-anchored adhesins expressed by most indigenous oral streptococci. Proteins sharing 30-40% overall amino acid sequence similarities with AgI/II-family proteins are also expressed by Streptococcus pyogenes. The S. pyogenes M28_Spy1325 polypeptide (designated AspA) displays an AgI/II primary structure, with alanine-rich (A) and proline-rich (P) repeats flanking a V region that is projected distal from the cell. In this study it is shown that AspA from serotype M28 S. pyogenes, when expressed on surrogate host Lactococcus lactis, confers binding to immobilized salivary agglutinin gp-340. This binding was blocked by antibodies to the AspA-VP region. In contrast, the N-terminal region of AspA was deficient in binding fluid-phase gp-340, and L. lactis cells expressing AspA were not agglutinated by gp-340. Deletion of the aspA gene from two different M28 strains of S. pyogenes abrogated their abilities to form biofilms on saliva-coated surfaces. In each mutant strain, biofilm formation was restored by trans complementation of the aspA deletion. In addition, expression of AspA protein on the surface of L. lactis conferred biofilm-forming ability. Taken collectively, the results provide evidence that AspA is a biofilm-associated adhesin that may function in host colonization by S. pyogenes.

Fig. 1

Pictorial representation of AgI/II proteins of (A) S. pyogenes serotype M28 (strain MGAS6180) and (B) S. gordonii DL1, showing the recombinant fragments generated in this study. Included are the aa residue numbers demarcating each of the protein fragments. Structural features are as follows: LS, leader sequence; N, N-terminal domain; A, alanine-rich repeats; V, variable region; P, proline-rich repeats; C, C-terminal domain; and CW, cell wall anchor.

Fig. 2

Western blots of recombinant protein fragments VP-AspA and NAV-SspB probed using anti-VP-AspA or anti-SpaP antibodies. Equivalent amounts of protein (100 ng) as determined by Bradford assay were applied to each lane. Proteins were also stained with Coomassie blue as a protein loading control.

Fig. 3

Metal ion analysis of recombinant protein fragments NAV-AspA (dark grey bars) and NAV-SspB (light grey bars) by ICP-OES using ICP multi-element standard solution IV (Merck) and 100 ng µl⁻¹ protein. Values are metal ion : protein ratios comparative to a buffer standard containing no protein. Error bars represent ± SD from three independent experiments. *P < 0.001 between samples as indicated.

Fig. 4

Binding of fluid-phase gp-340 by recombinant AspA or SspB protein fragments. SDS-PAGE patterns of proteins stained with Coomassie blue (left-side panels) and corresponding far-Western blots of (A) AspA-recombinant proteins AspA (NAVPC), NAV, VP and C, and (B) SspB-recombinant proteins SspB (NAVPC), NAV, VPC and C. Recombinant proteins (100–200 ng) were resolved by SDS-PAGE, blotted onto nitrocellulose and overlayered with 100 ng gp-340 ml⁻¹. Binding of gp-340 to the bands was detected by probing with mouse monoclonal gp-340 antibody followed by HRP-conjugated goat anti-mouse antibody.

Fig. 5

Interactions of L. lactis strains expressing AspA or SspB with immobilized gp-340 (50 ng). A. Binding of L. lactis MG1363 (pKS80) control cells, L. lactis (pKS80 sspB⁺) and L. lactis (pKS80 aspA⁺). Data represent the average ± SD of three independent experiments with triplicate samples. **P < 0.005 relative to L. lactis pKS80. B. Effect of dilutions of SpaP antiserum (grey fill) or rVP-AspA antiserum (black fill) on adherence of L. lactis (pKS80 aspA⁺) cells to gp-340. Control indicates no antiserum added. Input cell numbers in all experiments were 5 × 10⁷ cells well⁻¹. Error bars are ± SD of two independent experiments with duplicate samples. **P < 0.005, *P < 0.05, relative to no antiserum control.

Fig. 6

Aggregation of L. lactis strains expressing AspA or SspB in the presence of gp-340 or SRCRP2 peptide. Cells (10⁸ cells ml⁻¹) of each strain were incubated with 100 ng ml⁻¹ gp-340 or 100 ng ml⁻¹ SRCRP2 peptide, or in PBS alone, at 37°C for up to 5 h. Optical density (600 nm) was measured at intervals over 5 h and % aggregation was calculated from change in OD₆₀₀, corrected for OD₆₀₀ decrease in controls. Columns are L. lactis MG1363 (open), L. lactis (pKS80 aspA+) (grey fill) and L. lactis (pKS80 sspB+) (black fill). The data represent the average ± SD of three independent experiments with triplicate samples. **P < 0.005, *P < 0.05, relative to respective buffer only controls.

Fig. 7

Confocal laser scanning microscopy images and biomass measurements of 24 h biofilms formed on salivary pellicle-coated coverslips, comparing biofilm structures of (A) S. pyogenes MGAS6180 and (B) strain H360, and corresponding isogenic ΔaspA mutants and complemented strains ΔaspA (aspA⁺). Biomass data were obtained by crystal violet assay (A₅₉₅) of corresponding biofilms formed on coverslips. One unit is equivalent to the grid square side length. Data are averages ± SD of three independent experiments. **P < 0.005 relative to MGAS6180 (A) or H360 (B) wild-type strains.

Fig. 8

Biofilm formation on gp-340, saliva and polystyrene by (A) S. pyogenes MGAS6180 and (B) strain H360 (black fill), and corresponding isogenic ΔaspA mutants (no fill) and complemented strains ΔaspA (AspA+) (grey fill). Biomass data were obtained by crystal violet (A₅₉₅) assay. Values given represent mean ± SD of three biological replicates from an experiment repeated twice. **P < 0.01, *P < 0.05, relative to respective MGAS6180 or H360 wild-type strains.

Fig. 9

Confocal laser scanning microscopy images and corresponding biomass measurements of 24 h biofilms formed on salivary pellicle-coated coverslips of (A) wild-type L. lactis MG1363; (B) L. lactis (pKS80 aspA⁺); and (C) L. lactis (pKS80 sspB⁺). One unit is equivalent to the grid square side length. Biomass results (D) were obtained by crystal violet assay (A₅₉₅) of corresponding biofilms formed on coverslips. The biomass data are averages ± SD of three independent experiments. **P < 0.005, *P < 0.05, relative to L. lactis MG1363 wild-type.