Selective substitution of amino acids limits proteolytic cleavage and improves the bioactivity of an anti-biofilm peptide that targets the periodontal pathogen, Porphyromonas gingivalis

The interaction of the periodontal pathogen, Porphyromonas gingivalis, with oral streptococci such as Streptococcus gordonii precedes colonization of the subgingival pocket and represents a target for limiting P. gingivalis colonization of the oral cavity. Previous studies showed that a synthetic peptide (designated BAR) derived from the antigen I/II protein of S. gordonii was a potent competitive inhibitor of P. gingivalis adherence to S. gordonii and subsequent biofilm formation. Here we show that despite its inhibitory activity, BAR is rapidly degraded by intact P. gingivalis cells in vitro. However, in the presence of soluble Mfa protein, the P. gingivalis receptor for BAR, the peptide is protected from proteolytic degradation suggesting that the affinity of BAR for Mfa is higher than for P. gingivalis proteases. The rate of BAR degradation was reduced when the P. gingivalis lysine-specific gingipain was inhibited using the specific protease inhibitor, z-FKcK, or when the gene encoding the Lys-gingipain was inactivated. In addition, substituting d-Lys for l-Lys residues in BAR prevented degradation of the peptide when incubated with the Lys-gingipain and increased its specific adherence inhibitory activity in a S. gordonii–P. gingivalis dual species biofilm model. These results suggest that Lys-gingipain accounts in large part for P. gingivalis-mediated degradation of BAR and that more effective peptide inhibitors of P. gingivalis adherence to streptococci can be produced by introducing modifications that limit the susceptibility of BAR to the Lys-gingipain and other P. gingivalis associated proteases.

Research highlights▶ BAR peptide inhibits adherence of Porphyromonas gingivalis to Streptococcus gordonii. ▶ P. gingivalis-mediated proteolytic degradation of BAR occurs rapidly but BAR is protected from cleavage by its soluble receptor, the Mfa protein. ▶ Degradation of BAR peptide is primarily mediated by the lysine-specific gingipain of P. gingivalis. ▶ Substitution of d-Lys for l-Lys in BAR confers resistance to P. gingivalis-mediated proteolytic degradation. ▶ The d-Lys analog of BAR is a more potent inhibitor of P. gingivalis adherence to Streptococcus gordonii.