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. 2002 Feb;70(2):993-7.
doi: 10.1128/IAI.70.2.993-997.2002.

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

Yutaka Terao  1 , Shigetada Kawabata, Eiji Kunitomo, Ichiro Nakagawa, Shigeyuki Hamada
Affiliations

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

Yutaka Terao et al. Infect Immun. 2002 Feb.

Abstract

The lbp gene, which encodes a laminin-binding protein (Lbp) of Streptococcus pyogenes, was found in all S VSports手机版. 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. .

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Figures

FIG. 1.
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.
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.
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.
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.
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.
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