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: 10673635
• 负责人: Juhi Bagaitkar
• 金额: $ 53.38万
• 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/10673635
• 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; Osteitis; 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; 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; theories

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牙周膜中过度的、不受控制的蛋白水解活性驱动的。 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）， 齿垢密螺旋体、Tannerella denticola（Tf）和中间普雷沃氏菌分泌蛋白酶，这些蛋白酶有助于 炎症反应起始和传播。炎症无意中与释放 来自免疫的宿主蛋白酶，其不仅通过蛋白质降解增加组织破坏， 细胞外基质的成分和炎症的增强，但也是主要的执行者， 不可逆牙周组织损伤。另一方面，嗜中性粒细胞丝氨酸蛋白酶（NSP）是必需的， 中性粒细胞抗菌功能和控制炎症过程。在这方面，令人着迷的是， 产生一种脂蛋白，一种细菌丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），称为miropin。Miropin位于 Tf细胞表面有效地抑制多种多样特异性的丝氨酸和半胱氨酸蛋白酶， 来源，包括人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. Kgp产生的Lys特异性牙龈菌蛋白酶（Kgp）与Arg特异性牙龈菌蛋白酶（RgpA和RgpB）一起， 在几种动物模型中，牙龈卟啉单胞菌致病性的绝对必要的毒力因子 感染然而，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。 在鼠皮下腔室模型中，在共感染期间，以米罗平依赖性方式进行（我们的初步数据）。 基于这些结果，我们推测转铁蛋白衍生的miropin在病理生物学中起着双重的“阴阳”作用 牙周炎此外，我们推测，利用miropin独特的结构特征， 使用反应位点环内的至少3个不同活性位点来抑制不同特异性的蛋白酶， 将产生选择性特异性的supermiropin，靶向宿主或细菌来源的蛋白酶。 总的来说，这项提议的主要目的是阐明表达miropin的Tf在肿瘤细胞中的作用。 具体目的：（i）确定miropin在Tf-Pg共感染中对牙周炎的作用， 牙龈炎症和牙槽骨丢失（ABL）在小鼠口腔灌胃牙周炎模型;（ii）遗传 表达抑制多种宿主和牙龈卟啉菌蛋白酶的超级抗菌肽的工程化Tf突变株;（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;和 (iv)阐明宿主蛋白酶和牙龈卟啉菌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Pg和Tf之间的关系 靶向选择性的蛋白酶组。这一知识将创造新的视角，在治疗 牙周炎