Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein

Abstract

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.

FIG. 1

(A) Western blot analysis of the biotinylated P. gingivalis 33277 outer membrane extract coprecipitated with purified SspB protein. Lanes: 1, outer membrane extract only; 2, outer membrane extract coprecipitated with SspB protein; 3, SspB protein without outer membrane extract. (B) Western blot analysis of the biotinylated P. gingivalis 33277 outer membrane extract binding to E. faecalis strains. Lanes: 1, outer membrane extract only; 2, outer membrane extract reacted with E. faecalis EB5; 3: outer membrane extract reacted with E. faecalis 401.

FIG. 2

Western blot of P. gingivalis outer membrane extract reacted with antibodies to whole P. gingivalis cells (lane 1), the 100-kDa protein (lane 2), and the 70-kDa protein (lane 3).

FIG. 3

Dose-dependent inhibition of coadhesion between P. gingivalis and S. gordonii by 100-kDa antibodies or by 70-kDa control antibodies (Ab). The number of P. gingivalis cells bound to streptococci in the absence of antibodies was 4.1 × 10⁷ from an input cell number of 1 × 10⁸. Error bars represent standard errors of the means of three experiments.

FIG. 4

Arrangement and the sizes of the ORF containing SspB binding protein (ORF2) and the ORFs immediately upstream and downstream in strain W83. White areas indicate a noncoding region, and shaded areas indicate ORFs. Arrows indicate the positions and directions of the primers used in RT-PCR.

FIG. 5

RT-PCR of strain 33277 mRNA expressed from the ORF encoding the SspB-binding protein and the ORFs upstream and downstream. Lanes: 1, primers ORF1-1 and ORF2-2; 2, primers ORF2-1 and ORF2-2; 3, primers ORF2-1 and ORF3-2.