A T.fosythia-derived protease inhibitor in periodontal health and disease

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Chronic inflammation in periodontitis is driven in part by the excessive, uncontrolled proteolytic activity in the infected periodontium. Well recognized periodontal pathogens, including Porphyromonas gingivalis (Pg), Treponema denticola, Tannerella forsythia (Tf), and Prevotella intermedia, secrete proteases that contribute to initiation and propagation of inflammatory reaction. The inflammation is inadvertently associated with release of host proteases from immune, which not only add to tissue destruction by degradation of proteinaceous components of extracellular matrix and enhancement of inflammation, but are also the major executioners of irreversible periodontal tissue damage. On the other side, neutrophil serine proteases (NSPs) are essential for neutrophil antibacterial functions and control of inflammatory processes. In this respect it is fascinating that Tf produces a lipoprotein, a bacterial serpin (serine protease inhibitor) referred to as miropin. Miropin located on the Tf cell surface efficiently inhibits a broad range of serine and cysteine proteases of diverse specificities and origin, including human NSPs (elastase and cathepsin G), cysteine cathepsin L and bacterial enzymes, including Lys-specific gingipain (Kgp) produced by Pg. Kgp, together with Arg-specific gingipains (RgpA and RgpB), is absolutely essential virulence factor responsible for P. gingivalis pathogenicity in several animal models of infection. Nevertheless, it was surprising that wild-type Tf totally abolished Pg-induced morbidity and mortality in miropin-dependent manner during co-infection in a murine subcutaneous chamber model (our preliminary data). Based on these results we hypothesized that Tf-derived miropin plays a dual ‘yin–yang’ role in the pathobiology of periodontitis. Furthermore, we theorized that taking advantage of miropin unique structural features allowing it to inhibit proteases of different specificities using at least 3 different active sites within the reactive site loop we will produce supermiropins of selective specificities, targeting either host- or bacteria-derived proteases. Cumulatively, the main objective of this proposal is to unravel the role of miropin-expressing Tf in the pathobiology of periodontitis as outlined in Specific Aims: (i) determine effect of miropin in Tf-Pg co-infection on gingival inflammation and alveolar bone loss (ABL) in a mouse oral gavage periodontitis model; (ii) genetically engineer Tf mutant strains expressing a supermiropin that inhibits a wide range of host and gingipains; (iii) investigate effects of purified recombinant inhibitors and Tf strains expressing them on neutrophil functions; and (iv) elucidate the mechanisms underlying the roles of host proteases and gingipains in pathogenic interactions between Pg and Tf in oral, lung and chamber models of infection, using Tf strains expressing supermiropins targeting selective sets of proteases. This knowledge will create novel perspectives in the treatment of periodontitis.