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

牙周炎的慢性炎症在一定程度上是由牙周组织中过度的、不受控制的蛋白分解活动引起的 感染的牙周组织。公认的牙周病原体，包括牙龈卟啉单胞菌(PG)， 齿密螺旋体、连翘坦纳氏菌和中间普雷沃特氏菌分泌的蛋白酶有助于 炎症反应的启动和传播。炎症不经意间与释放 来自免疫的宿主蛋白酶，它不仅通过蛋白质的降解增加组织的破坏 细胞外基质的成分和炎症的增强，但也是主要的执行者 不可逆转的牙周组织损伤。另一方面，中性粒细胞丝氨酸蛋白酶(NSP)对于 中性粒细胞的抗菌功能和炎症过程的控制。在这方面，令人着迷的是， 产生一种脂蛋白，一种细菌丝氨酸(丝氨酸蛋白酶抑制剂)，称为米洛平。Miropin位于 转铁蛋白细胞表面可有效抑制多种不同特性的丝氨酸和半胱氨酸蛋白酶。 起源，包括人类NSP(弹性蛋白酶和组织蛋白酶G)、半胱氨酸组织蛋白酶L和细菌酶，包括 由PG产生的Lys特异性牙龈痛(KGP)。KGP与Arg特异性牙龈痛(RgpA和RgpB)一起，是 在几种牙周炎动物模型中致病的绝对必要毒力因子 感染。然而，令人惊讶的是，野生型转铁蛋白完全消除了PG诱导的发病率和死亡率 在小鼠皮下腔模型(我们的初步数据)中，合并感染期间的微罗平依赖方式。 根据这些结果，我们推测转铁蛋白衍生的微管蛋白在病理生物学中具有双重的阴阳作用。 牙周炎。此外，我们的理论是，利用微针独特的结构特征 它利用反应部位环内至少3个不同的活性部位来抑制不同特性的蛋白水解酶 将产生选择性特异性的超级微柱，针对宿主或细菌衍生的蛋白酶。 总的来说，这个提议的主要目标是解开表达miropin的转铁蛋白在细胞内的作用。 牙周炎的病理生物学：(I)确定米罗平在TF-PG混合感染中的作用 小鼠口腔灌胃牙周炎模型中牙周炎和牙槽骨丢失(ABL)；(Ii)遗传学 表达一种能抑制多种寄主和牙周疼痛的超级微蛋白的工程TF突变菌株；(Iii) 研究纯化的重组抑制物和表达它们的转铁蛋白对中性粒细胞功能的影响； (4)阐明宿主蛋白水解酶和牙周疼痛在致病相互作用中的作用机制 口腔、肺部和腔室感染模型中PG和Tf之间的关系，使用表达Supermiroins的Tf菌株 以选择性的蛋白水解酶为目标。这一知识将创造新的视角来治疗 牙周炎。

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.