KINETICS AND PROTEOLYTIC SIGNATURES OF HISTATIN 5 DEGRADATION IN SALIVA AND GINGIVAL CREVICULAR FLUID IN PERIODONTAL HEALTH AND GINGIVITIS

Periodontal disease progression centers on microbial dysbiosis and host defense compromise. Histatin 5, a key salivary antimicrobial protein, suffers proteolytic degradation across physiological and pathological states, yet its degradation kinetics and clinical relevance in periodontal disease remain poorly understood. This study investigates whether the histatin 5 degradation rate and mode, assessed through proteolytic fragment profiling (liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)) and degradation kinetics (Cationic polyacrylamide gel electrophoresis (PAGE) with band degradation quantification), can provide insights into periodontal disease activity in saliva and gingival crevicular fluid (GCF). Four objectives guided this research: (1) identifying oral bacteria (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus. mutans) capable of degrading histatin 5 in vitro and characterizing the resulting peptide fragments; (2) comparing histatin 5 degradation rate in pooled saliva (whole saliva supernatant (WSS)) and GCF from healthy (n=9) and gingivitis (n=15) subjects; (3) detecting the oral bacterium identified in vitro as capable of degrading histatin 5 in clinical samples using droplet digital PCR (ddPCR); and (4) comparing proteomic profiles of histatin 5 degradation between clinical and in vitro settings. In vitro, only P. gingivalis degraded histatin 5, producing 40 distinct fragments within 6 hours. Clinically, gingivitis accelerated histatin 5 degradation: saliva achieved complete breakdown by 4 hours (vs. 43% integrity in health), while GCF retained 78% intact protein in healthy samples at 48 hours but showed accelerated degradation in disease. Degradation rates in gingivitis were higher than health (25.38% vs. 4.13% band degradation loss at 0.5 hours), persisting even in P. gingivalis-negative samples (22.99% vs. 9.6%), indicating synergistic activity of microbial proteases and host-derived enzymes, amplified by the inflammatory microenvironment. P. gingivalis was detected in only 13.3% of gingivitis and 11.1% of healthy samples, challenging its presumed ubiquity and supporting community-driven dysbiosis. Proteomic analysis identified significantly greater fragment diversity in gingivitis (93 and 75 fragments in absence and presence of P. gingivalis) versus in vitro degradation by P. gingivalis alone (40 fragments). This amplified complexity arose from synergistic bacterial-host proteolytic activity in the clinical environment. Proteomic analysis further revealed distinct differences in the composition and abundance of proteolytic enzymes between healthy and gingivitis groups, originating from both human and microbial sources. Gingivitis samples exhibited a higher abundance of proteolytic enzymes, reinforcing the concept of an active proteolytic state driven by dysbiotic inflammation. While fragment profiles partially mirrored in vitro patterns, gingivitis exhibited unique fragments. Conversely, healthy samples retained protective motifs (e.g., HEKHH domain). This study presents gingivitis as an active proteolytic state where synergistic microbial-host enzyme activity accelerates histatin 5 degradation. It establishes the degradation kinetics and modes of histatin 5 in both gingivitis and periodontal health, revealing distinct fragment and rate profiles in saliva and gingival crevicular fluid (GCF) that reflect periodontal disease activity. These proteolytic signatures serve as direct indicators of dysbiotic inflammation.