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

一种源自 T.fosythia 的蛋白酶抑制剂对牙周健康和疾病的影响

• 批准号: 10279103
• 负责人: Juhi Bagaitkar
• 金额: $ 54.69万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-12 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10279103
• 关键词: Active Sites; Affect; Alveolar Bone Loss; Animal Model; Anti-Bacterial Agents; Automobile Driving; Bacteria; Binding Sites; Biological Response Modifiers; Blood coagulation; Caspase; Cathepsin G; Cathepsin L; Cell surface; Cells; Chronic; Clinical; Communities; Complex; Cysteine; Data; Dental Plaque; Disease; Elastases; Enzymes; Event; Extracellular Matrix; Extracellular Matrix Proteins; Forsythia; Genetic Engineering; Gingiva; Goals; Gram-Negative Anaerobic Bacteria; Growth Factor; Health; Human; Immune; Infection; Inflammation; Inflammatory; Knowledge; Light; Lipoprotein (a); Lung; Lys-gingipain; Modeling; Morbidity - disease rate; Mus; Names; Nature; Nutrient; Oral; Organism; Outcome; Pathogenesis; Pathogenicity; Peptide Hydrolases; Periodontal Diseases; Periodontal Pocket; Periodontitis; Periodontium; Play; Porphyromonas gingivalis; Prevention; Prevotella intermedia; Process; Prokaryotic Cells; Protease Inhibitor; Reaction; Recombinants; Research; Resolution; Role; Serine Protease; Serine Proteinase Inhibitors; Serpins; Side; Signal Transduction; Site; Specificity; Structure; Testing; Thrombus; Tissues; Treponema denticola; Virulence; Virulence Factors; Yin-Yang; base; chronic inflammatory disease; co-infection; controlled release; design; dysbiosis; fascinate; gingipain; in vivo Model; inhibitor/antagonist; member; mortality; mutant; neutrophil; novel; pathobiont; pathogen; periodontopathogen; receptor; scaffold; subcutaneous; subgingival biofilm; subgingival microbiome

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.

牙周炎中的慢性炎症部分是由过量的，不受控制的蛋白水解活性驱动的 感染的牙周。公认的牙周病原体，包括牙龈卟啉单胞菌（PG）， treponema denticola，Tannerella forsythia（TF）和Prevotella Intermedia，秘密蛋白酶有助于 炎症反应的引发和传播。炎症与释放无意间有关 免疫的宿主蛋白酶不仅会因蛋白质降解而增加组织破坏 细胞外基质的组成部分和炎症的增强，但也是主要执行者 不可逆的牙周组织损伤。另一方面，中性粒细胞丝氨酸蛋白酶（NSP）对于 中性粒细胞抗菌功能和炎症过程的控制。在这方面，TF令人着迷 产生脂蛋白，脂蛋白，一种称为miropin的细菌丝蛋白（丝氨酸蛋白抑制剂）。 Miropin位于 TF细胞表面有效地抑制了潜水规格的广泛丝氨酸和半胱氨酸蛋白酶，以及 起源，包括人NSP（弹性酶和组织蛋白酶G），半胱氨酸组织蛋白酶L和细菌，包括 由Pg产生的Lys特异性金刚蛋白酶（KGP）。 kgp以及特定于ARG特异性的gingipain（RGPA和RGPB）是 在几种动物模型中，绝对必要的病毒因素负责牙龈疟原虫的致病性 感染。然而，令人惊讶的是，野生型TF完全废除了PG引起的发病率和死亡率 在鼠皮下腔室模型（我们的初步数据）中共感染期间，依赖Miropin的方式。 基于这些结果，我们假设TF衍生的Miropin在病理生物学中起双重“ Yin – yang”角色 牙周炎。此外，我们从理论上说，利用米罗蛋白独特的结构特征允许 它可以使用反应性位点环内的至少3个不同的活性位点抑制不同规格的蛋白酶 将产生选择性规格的超霉素，靶向宿主或细菌衍生的蛋白酶。 累积地，该提案的主要目的是揭示表达miropin的TF的作用 特定目的中概述的牙周炎病理生物学：（i）确定miropin在TF-PG共感染中的影响 小鼠口服牙周炎模型中的牙龈炎症和肺泡骨质流失（ABL）； （ii）一般 工程师TF突变菌株表达了一种超霉素，可抑制各种宿主和金aipans； （iii） 研究纯化的重组抑制剂和在中性粒细胞功能上表达它们的TF菌株的作用；和 （iv）阐明宿主蛋白酶和金ai在致病相互作用中的作用的机制 使用表达超霉素的TF菌株在口腔，肺和腔室模型中的PG和TF之间 靶向选择性蛋白酶。这些知识将在治疗中创造新的观点 牙周炎。