ABS 072: Identification of Gingipain Inhibitors From the Oral Microbiome

Linda Kebichi ¹ , Shikha Sharma ¹ , Akintunde Emiola ¹

¹ NIDCR, National Institutes of Health

Van Wickle (2025) Volume 1, ABS 072

Introduction: Periodontitis is a serious infection of the gums characterized by severe inflammation and caused by bacteria that have accumulated on the teeth and gums. Porphyromonas gingivalis is a key pathogenic bacteria involved in periodontitis which secretes proteases called gingipains that are key virulence factors in the progression of periodontal disease. Thus, finding inhibitors of such proteases serves as an area for the discovery of novel therapeutics for periodontitis. This study examines the inhibitory effect of proteins and metabolites secreted by several species of streptococci. A fluorescence assay was used to measure the ability of an inhibitor to block RGP activity. Lower fluorescence values were correlated with greater inhibition of RGP activity, as the fluorescent peptide only fluoresces when cleaved by the RGP protease. A mixture of proteins and metabolites secreted by Streptococcus gordonii DL1 was found to inhibit RGP activity. This project sought to further characterize and isolate the inhibitor for future sequencing and identification. The inhibitor exhibited reduced performance when heat-treated at higher temperatures, indicating that it is a protein. Following a chloroform-methanol extraction, the interphase layer was also found to have the greatest inhibitory effect, indicating that the inhibitor is a small peptide. Further purification and isolation methods including size exclusion filtration should be performed prior to sequencing and identification of the inhibitor. Since this peptide has been isolated from S. gordonii, which is part of a healthy oral microbial community, it can be used in the future as a therapy against periodontitis while maintaining homeostasis of the oral microbiome.

Methods: This experiment aims to extract an RGP inhibitor from streptococci and assess its ability to inhibit RGP activity from P. gingivalis. Effective inhibition will prevent cleavage of a commercial fluorescent peptide, resulting in reduced fluorescence.

Inhibitor extraction from streptococci:
Streptococci are grown in media at 37°C, centrifuged (5 min, 42,000 g), and 3 mL of supernatant is filtered (0.22 filter). After adding 3 mL butanol, the mixture is shaken (30 min, 28°C). 1 mL of the organic phase is dried in a speed vacuum and resuspended in 50 μL PBS.

P. gingivalis protein extraction:
Cultured anaerobically, P. gingivalis is centrifuged (10 mL, 42,000 g). Supernatant (3 mL) is filtered (10K), combined with 1 mL PBS, and centrifuged. 100–200 μL is collected, avoiding impurities. A chloroform-methanol protein extraction is performed, then the pellet is air dried and resuspended in 100 μL PBS.

Inhibitor assay:
Combine 170 μL peptide buffer (1:1 commercial fluorescent peptide : peptide buffer), 30 μL P. gingivalis sample, and 6 μL inhibitor. Incubate (37°C, 30 min) and measure fluorescence.

Results and Discussion: In this project, S. gordonii DL1 was found to secrete an agent that inhibits RGP, reducing fluorescence when a peptide substrate was treated with it. Heat sensitivity tests suggest the inhibitor is a protein, as its activity declined after treatment at 50°C. A chloroform-methanol extraction showed the inhibitor was most concentrated in the interphase, indicating it is likely a small peptide. Future steps include size-based filtration and sequencing. This inhibitor, naturally secreted by S. gordonii, may serve as a targeted therapy for periodontal disease without harming beneficial bacteria in the oral microbiome.