Novel laminin-binding protein of Streptococcus pyogenes, Lbp, is involved in adhesion to epithelial cells

Abstract

The lbp gene, which encodes a laminin-binding protein (Lbp) of Streptococcus pyogenes, was found in all S. pyogenes M types. An Lbp-deficient mutant showed a significantly lower efficiency of adhesion to HEp-2 cells than did the wild-type strain. These results indicate that Lbp is one of the important S. pyogenes adhesins.

FIG. 1.

Chromosomal DNAs from group A (GAS), B (GBS), C (GCS), D (GDS), and G (GGS) and oral streptococci were purified with a Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.). DNA was digested with EcoRI and subjected to Southern hybridization analysis. The lbp gene was employed as a probe.

FIG. 2.

(A) Construction of the Lbp expression plasmid vector. The fragment containing codons 17 to 306 of the lbp gene was amplified from the SSI-9 (M1) genome as a template and inserted into pGEX-6P-1 (Amersham Pharmacia Biotech), which was then named pYT1097. The recombinant protein was lacking a signal peptide in the N-terminal region. (B) rLbp was purified by single-step affinity chromatography and immobilized on a PVDF membrane. (a) Coomassie brilliant blue staining. (b) Biotinylated human Lm solution (100 μg/ml) was added to the membrane. The reaction was developed with horseradish peroxidase (HRP)-labeled streptavidin. (c) Only HRP-labeled streptavidin was added. (C) Western blot analysis with rabbit anti-Lbp serum and urea extracts of S. pyogenes. Proteins were extracted from M1 (SSI-9), M3 (SSI-1), and M12 (#42) with 8 M urea (lanes 1 to 3, respectively). Samples were subjected to SDS-PAGE and then transferred to a PVDF membrane. (a) Coomassie brilliant blue staining. (b) Western blotting using rabbit anti-Lbp serum. Lane M contained molecular size markers.

FIG. 2.

(A) Construction of the Lbp expression plasmid vector. The fragment containing codons 17 to 306 of the lbp gene was amplified from the SSI-9 (M1) genome as a template and inserted into pGEX-6P-1 (Amersham Pharmacia Biotech), which was then named pYT1097. The recombinant protein was lacking a signal peptide in the N-terminal region. (B) rLbp was purified by single-step affinity chromatography and immobilized on a PVDF membrane. (a) Coomassie brilliant blue staining. (b) Biotinylated human Lm solution (100 μg/ml) was added to the membrane. The reaction was developed with horseradish peroxidase (HRP)-labeled streptavidin. (c) Only HRP-labeled streptavidin was added. (C) Western blot analysis with rabbit anti-Lbp serum and urea extracts of S. pyogenes. Proteins were extracted from M1 (SSI-9), M3 (SSI-1), and M12 (#42) with 8 M urea (lanes 1 to 3, respectively). Samples were subjected to SDS-PAGE and then transferred to a PVDF membrane. (a) Coomassie brilliant blue staining. (b) Western blotting using rabbit anti-Lbp serum. Lane M contained molecular size markers.

FIG. 3.

(A) Targeted mutagenesis of the lbp gene in S. pyogenes SSI-9. pYT1088 contains an internal fragment of lbp and a kanamycin resistance-encoding gene (aphA3), whereas mutant TR-7 was produced by single-crossover recombination. (B) Lm-binding assay with GST (lane 1), rLbp (lane 2), and 8 M urea extracts of strains SSI-9 (wild type, lane 3) and TR-7 (Δlbp, lane 4). Samples were separated by SDS-PAGE and transferred to a PVDF membrane. (a) Coomassie brilliant blue staining. (b) Biotinylated human Lm solution (100 μg/ml) and HRP-labeled streptavidin.

FIG. 4.

Effects of Lbp on bacterial adhesion to and invasion of HEp-2 cells. Bacteria were suspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and then used to infect HEp-2 monolayers. HEp-2 cells were grown in a 24-well plate at a density of 5 × 10⁴ per well. Approximately 10⁷ bacteria were added to each well (multiplicity of infection, 1:200), and the plate was incubated for 3 h at 37°C. To determine bacterial adhesion, cells were washed with DMEM, lysed with 1 ml of sterile distilled water, and then plated to determine the number that were adhered to or invaded by S. pyogenes. (A) The percentage of adhesion or invasion was calculated as follows: (no. of CFU adhered to or invaded/total no. of CFU in inoculum) × 100. (B) Prior to bacterial invasion, cells were washed and incubated for 1 h in DMEM containing gentamicin (100 μg/ml) and penicillin (10 U/ml). Cells were washed, lysed, and plated for counting of those invaded by S. pyogenes. The percentage of invasion was calculated as follows: (no. of CFU invaded/total no. of CFU in inoculum) × 100. The results shown are the means ± the standard errors of the means of six wells (three experiments were performed in triplicate). Statistical analysis was performed with a nonparametric Mann-Whitney U test. All conclusions were based on a significance level of P < 0.005.