BACTERIAL PROTEINASES IN PERIODONTAL DISEASE

描述（由申请人提供）：牙周炎被分类为微生物群转移疾病，其中具有良好口腔健康特征的非常丰富和多样的口腔植物群被几种革兰氏阴性厌氧菌种所支配，其中牙龈卟啉单胞菌、嗜酸坦那菌和齿垢密螺旋体构成了通常与疾病的发展和进展相关的所谓的微生物“红色复合体”。这三种细菌和继发性牙周病病原体中间普雷沃氏菌的共同结果是它们产生蛋白水解酶。牙龈卟啉单胞菌和T.已知齿垢使组织稳态失调和/或使防御性炎症反应受挫，因此，显著地促进了受感染牙周组织的病理变化。相比之下，实际上对T.虽然已经充分证明来自前一种生物体的蛋白酶在体内产生并且它们的活性与牙周破坏相关。在这里，我们假设T. P. intermedia（interpains）通过严格控制的宿主蛋白水解系统的失调促进牙周组织损伤。此外，我们认为，结合牙周病原体产生的局部蛋白水解活性可能是协同牙周组织损伤的主要机制，并有助于相互生存的红色复合菌。最有趣的是，我们发现牙龈卟啉单胞菌和嗜热链球菌的胰蛋白酶和间质蛋白酶以及许多其他公认的毒力因子。该菌与牙龈卟啉菌蛋白酶共享保守的C-末端结构域。我们已经表明，这个结构域，连同一个独特的周质蛋白和一个独特的外膜转位蛋白在这些物种中保守是必不可少的牙龈菌蛋白酶分泌。这意味着一种新的细菌蛋白质输出系统是由牙周病原体分泌的一些确认和推定的毒力因子。因此，本课题的具体目标如下：1）利用一系列生物化学和分子生物学方法研究牙龈卟啉菌蛋白酶通过牙龈卟啉菌外膜转运的机制，并证实这一独特的分泌途径在牙龈卟啉菌和牙龈卟啉菌中的存在。（2）对T.为了确定来自牙龈卟啉单胞菌、中间卟啉单胞菌和中间卟啉单胞菌的蛋白酶的互补或协同作用，特别是关于它们与牙龈卟啉单胞菌蛋白酶活性互补和/或协同作用的能力，以及3）确定来自牙龈卟啉单胞菌、中间卟啉单胞菌和中间卟啉单胞菌的蛋白酶的互补或协同作用。使用鼠室模型，在单一微生物和混合微生物感染中，使用P. 公共卫生相关性：这些研究结果不仅阐明了细菌蛋白酶在混合感染中的协同作用，而且还阐明了牙龈卟啉单胞菌、T. P. intermedia.这种见解可能使抗体或化学化合物的设计，以阻止出口的多种毒力因子从主要牙周病原体，这在长期来看，可能会彻底改变牙周病的治疗和预防。

DESCRIPTION (provided by applicant): Periodontitis is classified as a microbiota shift disease, whereby a very rich and diverse commensal flora characteristic for good oral health becomes dominated by several Gram-negative, anaerobic bacterial species, among which, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola constitute the so called "red complex" of microorganisms frequently associated with the development and progression of the disease. The common consequence of these three bacteria and a secondary periodontopathogen, Prevotella intermedia, is their production of proteolytic enzymes. Proteases of P. gingivalis and T. denticola are known to dysregulate tissue homeostasis and/or to frustrate defensive inflammatory responses, thus, significantly contributing to pathological changes in the infected periodontium. In contrast, virtually nothing is known about proteases of T. forsythia and P. intermedia although it has been well documented that proteases from the former organism are produced in vivo and their activity correlates with periodontal destruction. Here, we postulate that novel proteases produced by T. forsythia (forsypsin) and P. intermedia (interpains) promote periodontal tissue damage by deregulation of tightly controlled host proteolytic systems. Furthermore, we suggest that the combined proteolytic activity produced locally by periodontopathogens may be a primary mechanism for synergistic periodontal tissue damage and contributes to mutual survival of the red complex bacteria. Most interestingly, we have found that forsypsin and interpains, as well as many other acknowledged virulence factors of P. gingivalis and T. forsythia share with gingipains a conserved C-terminal domain. We have shown that this domain, together with a unique periplasmic protein and a unique outer membrane translocon protein conserved in these species is essential for gingipain secretion. This implies that a novel bacterial protein export system is employed by periodontopathogens to secrete a number of confirmed and putative virulence factors. For these reasons, the specific aims of this project are as follows: 1) to use an array of biochemical and molecular biological methods to investigate the mechanism of gingipain translocation through the outer membrane of P. gingivalis and confirm the presence of this unique secretion pathway in both T. forsythia and P. intermedia; 2) to characterize novel proteases from T. forsythia and P. intermedia, especially with regard to their ability to complement and/or synergize with the gingipain activity, and 3) to determine the complementary or synergistic role of proteases from P. gingivalis, T. forsythia and P. intermedia for in vivo proliferation and dissemination of bacteria in monomicrobial and mixed microbial infections using a murine chamber model. PUBLIC HEALTH RELEVANCE: The results of these proposed studies should not only illuminate the synergistic role of bacterial proteases in mixed infections but also shed light on a novel pathway for the secretion of virulence factors unique to P. gingivalis, T. forsythia and P. intermedia. This insight may enable the design of antibodies or chemical compounds to block the export of multiple virulence factors from major periodontopathogens, which in the long term, may revolutionize treatment and prophylaxis of periodontal disease.