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

基本信息

批准号：
10447716
负责人：
Juhi Bagaitkar
金额：
$ 52.85万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
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/10447716
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 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 member mortality mutant neutrophil novel pathobiont pathogen periodontopathogen receptor scaffold subcutaneous subgingival biofilm subgingival microbiome

项目摘要

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.

牙周炎的慢性炎症部分是由牙周组织中过度的、不受控制的蛋白水解活性引起的。感染的牙周组织。众所周知的牙周病原体，包括牙龈卟啉单胞菌（Pg），齿垢密螺旋体、连翘坦纳菌 (Tf) 和中间普雷沃菌分泌的蛋白酶有助于炎症反应的发生和传播无意中与释放的物质相关。来自免疫的宿主蛋白酶，这不仅通过蛋白质降解而增加组织破坏细胞外基质的成分和炎症的增强，但也是主要的刽子手另一方面，中性粒细胞丝氨酸蛋白酶（NSP）对于牙周组织损伤至关重要。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Tf。产生脂蛋白，一种细菌丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），称为米罗平（Miropin）。 Tf 细胞表面有效抑制多种具有不同特异性的丝氨酸和半胱氨酸蛋白酶，来源，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 Pg 产生的 Lys 特异性牙龈蛋白酶 (Kgp) 与 Arg 特异性牙龈蛋白酶（RgpA 和 RgpB）一起，是牙龈卟啉单胞菌在几种动物模型中致病性的绝对必需毒力因子然而，令人惊讶的是，野生型 Tf 完全消除了 Pg 诱导的发病率和死亡率。在小鼠皮下室模型中的共感染过程中，米罗平依赖的方式（我们的初步数据）。基于这些结果，我们发现 Tf 衍生的 miropin 在病理生物学中发挥双重“阴阳”作用此外，我们推测利用 miropin 独特的结构特征可以预防牙周炎。它使用反应位点环内至少 3 个不同的活性位点来抑制不同特异性的蛋白酶，我们将产生具有选择性特异性的 supermiropins，针对宿主或细菌衍生的蛋白酶。总的来说，该提案的主要目标是阐明表达 miropin 的 Tf 在具体目标中概述的牙周炎病理学：(i) 确定 Miropin 在 Tf-Pg 共感染中对牙周炎的影响小鼠口腔灌胃牙周炎模型中的牙龈炎症和牙槽骨丢失（ABL）；（ii）遗传；工程化表达 supermiropin 的 Tf 突变株，可抑制多种宿主和牙龈蛋白酶 (iii) 研究纯化的重组抑制剂和表达它们的 Tf 菌株对中性粒细胞功能的影响； (iv) 阐明宿主蛋白酶和牙龈蛋白酶在致病相互作用中的作用机制使用表达 supermiropins 的 Tf 菌株，在口腔、肺和腔室感染模型中比较 Pg 和 Tf 之间的关系针对选择性蛋白酶组的这些知识将为治疗创造新的视角。牙周炎。