Fluorescence Turn-On Sensor for Gingipains: Selective Detection of Porphyromonas gingivalis in Periodontal Disease

Porphyromonas gingivalis (P.g.), a key pathogen in periodontal disease, plays a critical role in bacterial invasion and is increasingly implicated in systemic conditions, including cardiovascular and neurodegenerative diseases. Its major virulence factors, gingipains, are cysteine proteases essential for pathogenicity. In this study, we introduce Gingitracker, an activity-based fluorescence turn-on sensor for the selective detection of gingipains. Upon covalent binding to the cysteine residue in the active sites of gingipains, Gingitracker undergoes a planar conformational change via the restriction of intramolecular rotation, leading to a significant increase in fluorescence (ΦKgp = 0.54, ΦRgpB = 0.35). In contrast, interactions with control proteases such as mammalian calpain (ΦCalpain = 0.04) and trypsin (ΦTrypsin = 0.03) result in negligible fluorescence, demonstrating Gingitracker's high specificity for gingipains. Gingitracker effectively detects active gingipains in dental plaque samples from periodontitis patients, showing strong correlations with anti-gingipain signals across independent samples (r = 0.73-0.83). These results establish Gingitracker as a sensitive activity-based tool for detecting active gingipains in bacterial and clinical periodontal samples.