BACTERIAL PROTEINASES IN PERIODONTAL DISEASE

描述(申请人提供)：牙周炎被归类为一种微生物区系移位性疾病，其良好的口腔健康的特征是非常丰富和多样化的共生菌群，由几种革兰氏阴性厌氧细菌主导，其中牙龈卟啉单胞菌、连翘和齿密螺旋体构成了与疾病的发生和发展频繁相关的所谓的微生物的“红色复合体”。这三种细菌和第二种牙周病原体中间普雷沃特氏菌的共同后果是它们产生蛋白水解酶。牙龈假单胞菌和齿假单胞菌的蛋白水解酶可调节组织内环境平衡和/或抑制防御性炎症反应，从而显著促进感染牙周组织的病理变化。相反，对连翘和中间连翘的蛋白酶几乎一无所知，尽管已经有充分的证据表明，来自前一种生物的蛋白酶是在体内产生的，它们的活性与牙周破坏有关。在这里，我们假设由连翘(连翘蛋白酶)和中间连翘(中间痛)产生的新的蛋白水解酶通过放松严格控制的宿主蛋白分解系统来促进牙周组织的损伤。此外，我们认为牙周病原体局部产生的联合蛋白分解活性可能是协同牙周组织损伤的主要机制，并有助于红色复合细菌的相互生存。最有趣的是，我们发现连翘蛋白酶和InterPain，以及许多已知的牙龈假单胞菌和连翘的毒力因子与牙龈痛共享一个保守的C-末端结构域。我们已经证明，这个结构域与一个独特的周质蛋白和一个在这些物种中保守的独特的外膜转位蛋白一起，对牙龈痛的分泌是必不可少的。这意味着一种新的细菌蛋白输出系统被牙周病原体利用来分泌一些已证实的和推测的毒力因子。因此，本项目的具体目标如下：1)利用多种生物化学和分子生物学方法研究牙周疼痛通过牙龈假单胞菌外膜转运的机制，并证实这一独特的分泌途径在连翘和中间连翘中均存在；2)鉴定连翘和中间连翘的新型蛋白水解酶，特别是它们与牙周疼痛活性的互补和/或协同作用的能力，以及3)使用小鼠小室模型来确定牙龈连翘、连翘和中间假单胞菌的蛋白酶在单一微生物和混合微生物感染中对细菌体内增殖和传播的互补或协同作用。 公共卫生相关性：这些拟议的研究结果不仅应该阐明细菌蛋白水解酶在混合感染中的协同作用，而且还应该阐明一种新的途径，分泌牙龈假单胞菌、连翘和中间假单胞菌特有的毒力因子。这一见解可能会使抗体或化合物的设计能够阻止主要牙周病原体的多种毒力因子的输出，从长远来看，这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.