BACTERIAL PROTEINASES IN PERIODONTAL DISEASE

描述（由申请人提供）：牙周炎被归类为一种微生物群转移疾病，具有良好口腔健康特征的非常丰富多样的共生菌群被几种革兰氏阴性厌氧菌所主导，其中牙龈卟啉单胞菌、连珠Tannerella forsythia和密螺旋体denticola构成了所谓的“红色复合体”微生物，通常与疾病的发生和进展有关。这三种细菌和一种继发性牙周病原中间普氏菌的共同后果是它们产生蛋白水解酶。已知牙龈假单胞菌和齿牙假单胞菌的蛋白酶会失调组织稳态和/或阻碍防御性炎症反应，因此，在感染的牙周组织中显著促进病理变化。相比之下，我们对连翘和中叶连翘的蛋白酶几乎一无所知，尽管已有文献证明前者的蛋白酶是在体内产生的，而且它们的活性与牙周破坏有关。在这里，我们假设连翘T. forsythia （forsypsin）和中间媒介P. intermedia （interpains）产生的新蛋白酶通过解除严格控制的宿主蛋白水解系统来促进牙周组织损伤。此外，我们认为牙周病病原体局部产生的联合蛋白水解活性可能是协同牙周组织损伤的主要机制，并有助于红色复合体细菌的相互生存。最有趣的是，我们发现连翘素和interpains以及许多其他已知的牙龈卟啉和连翘的毒力因子与gingipains共享一个保守的c -末端结构域。我们已经证明，这个结构域以及在这些物种中保守的独特的质周蛋白和独特的外膜转位蛋白对于牙龈蛋白酶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病原体用来分泌一些已证实和推测的毒力因子。因此，本课题的具体目的如下：1)利用一系列生化和分子生物学方法，研究牙龈蛋白酶通过牙龈外膜转运的机制，并证实这一独特的分泌途径在连翘和中叶连翘中都存在；2)对连翘和中间叶连翘的新蛋白酶进行表征，特别是它们与牙龈蛋白酶活性的互补和/或协同作用；3)利用小鼠室模型确定牙龈连翘、连翘和中间叶连翘的蛋白酶在单微生物和混合微生物感染中对细菌的体内增殖和传播的互补或协同作用。

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.