Structural dissection and in vivo effectiveness of a peptide inhibitor of Porphyromonas gingivalis adherence to Streptococcus gordonii

Abstract

The interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm by Porphyromonas gingivalis. We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interaction in vitro. Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reduces P. gingivalis colonization and alveolar bone loss in vivo in a murine model of periodontitis. Substitution of Gln for Pro(1171) or Glu(1168) increased the α-helicity of BAR and reduced its inhibitory activity in vitro by 10-fold and 2-fold, respectively. To determine if BAR prevents P. gingivalis infection in vivo, mice were first infected with Streptococcus gordonii and then challenged with P. gingivalis in the absence and presence of BAR. Animals that were infected with either 10(9) CFU of S. gordonii DL-1 or 10(7) CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10(9) CFU of S. gordonii followed by 10(7) CFU of P. gingivalis induced significantly greater bone loss (P < 0.01) than sham infection or infection of mice with either organism alone. S. gordonii-infected mice that were subsequently challenged with 10(7) CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity by P. gingivalis.

FIG. 1.

Secondary structure of BAR and BAR-15. (A) Secondary structure predictions of BAR and BAR-15 indicate that substitution of Gln for Pro¹¹⁷¹ increases the α-helical content of BAR-15. (B) Circular dichroism spectra of BAR (triangles) and BAR-15 (squares) show increased negative ellipticity for BAR-15 at the λ of 222 nm, consistent with increased α-helical structure.

FIG. 2.

(A) S. gordonii colonization of the murine oral cavity. Successful infection and establishment of S. gordonii DL-1 was assessed by PCR using gtfG-specific primers. Microbial samples obtained from each of the sham-infected animals 1 week after the last round of infection (lanes 2 to 7) did not generate the 440-bp gtfG amplicon, whereas samples obtained at the same time point from S. gordonii-infected mice (lanes 8 to 11 and 13 to 16) each produced the gtfG amplicon. Lanes 1 and 12 contain DNA size markers, and lane 17 represents a no-template negative-control reaction. (B) BAR does not affect S. gordonii colonization of the mouse oral cavity. Murine oral plaque samples were analyzed by PCR for the presence of S. gordonii as described above. Lane 1, DNA size markers; lanes 2 to 4, sham-infected mice; lanes 5 to 7, mice infected with S. gordonii; lanes 8 to 10, mice infected with S. gordonii in the presence of 3.4 μM BAR peptide; lane 11, S. gordonii genomic DNA positive control; and lane 12, no-template negative control. (C) PCRs using universal primers for the 5S rRNA gene indicated that all the plaque samples contained bacterial DNA. Lane 1, DNA size markers, lanes 2 to 4, sham-infected mice; lanes 5 to 7, mice infected with S. gordonii; lanes 8 to 10, mice infected with S. gordonii in the presence of 3.4 μM BAR peptide; lane 11, S. gordonii genomic DNA positive control; and lane 12, no-template negative control.

FIG. 3.

BAR treatment reduces alveolar bone loss in P. gingivalis-infected mice. Animals that were infected with either 10⁹ CFU of S. gordonii DL-1 or 10⁷ CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10⁹ CFU of S. gordonii followed by 10⁷ CFU of P. gingivalis induced significantly greater bone loss (*, P < 0.01) than sham-infected animals or mice infected with either organism alone. S. gordonii-infected mice that were subsequently challenged with 10⁷ CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to levels in sham-infected animals, suggesting that BAR inhibited adherence of P. gingivalis to streptococci in the oral cavity.

FIG. 4.

BAR is not toxic to human cells. (A) Hemolytic activity of BAR and BAR-17 was assessed by incubating sheep (gray bars) and human (black bars) erythrocytes with 3.4 μM and 34.0 μM BAR peptide for 180 min at 37°C. As a positive control, RBCs were lysed by incubation in distilled H₂O. As a negative control, RBCs were incubated in FBS buffer without BAR peptide. No significant hemoglobin release was observed from peptide-treated cells relative to H₂O-incubated samples (*, P < 0.001). (B) Peptide-induced necrosis was assessed by incubating OBA-9 oral epithelial cells with 3.4 μM or 34.0 μM BAR or BAR-17 as described in Materials and Methods. Compared with the staurosporine-treated positive-control reaction, no significant release of lactate dehydrogenase (*, P < 0.001) was measured from the peptide-treated or medium-treated cells. (C) Determination of total ATP content in BAR- or BAR-17-treated OBA-9 cells. ATP levels in peptide-treated cells did not differ significantly from levels in cells treated with medium alone, whereas ATP levels in the staurosporine-treated control cells was significantly lower (*, P < 0.001), suggesting that neither peptide significantly affects cell viability.

FIG. 5.

BAR does not induce apoptosis in HGEC. Apoptosis was determined using a TUNEL assay. Incubation of HGEC with BAR (A) did not induce apoptosis, and peptide-treated cells exhibited staining similar to cells incubated with medium only (B) or transferase-free buffer negative controls (C). DNase-treated (D) or staurosporine-treated (E) cells were TUNEL positive, as indicated by the green staining of the nuclei, suggesting that DNA cleavage and apoptosis occurred. All cell samples were counterstained with phalloidin (red). Em, emission wavelength.