Characterization of Treponema Denticola-Mediated Interactions With Periodontal Ligament Cells Leading to Persistent Host Cell/Tissue Destruction

项目总结/摘要 牙周病的特征在于构成牙周组织的硬组织和软组织的破坏。 牙周组织这种破坏转化为细胞外基质（ECM）的降解， 由细菌蛋白酶、宿主来源的基质金属蛋白酶（MMP）和其它蛋白酶介导 由宿主组织和免疫细胞释放。此外，致病微生物是牙周炎的催化剂， 疾病发病机制，因为微生物组的生态失调的发展增强了持续的免疫应答。 反应这些细菌病原体与宿主组织相互作用，从而触发不利的细胞功能， 包括增强的免疫反应、组织破坏和组织迁移。其中一个与细菌有关， 口腔螺旋体、齿垢密螺旋体（T.牙周炎或Td）与牙周病高度相关（5）。 我们已经证明了一种叫做牙本质素的Td外膜蛋白复合物， 牙周膜（PDL）细胞中MMP-2原的活化（26，27）。接触牙菌肽也会引发 PDL细胞中活性MMP-2的激活剂和效应物的表达水平。尽管取得了这些进展， 已知牙本质素诱导MMP-2活化或PDL细胞病变行为导致疾病的机制，或 潜在的治疗方法。对抗这些相互作用的一种可能的治疗剂是乳链菌肽。乳酸链球菌素是一种天然存在的 细菌素，广泛使用的食品防腐剂，和益生菌剂天然制成的细菌物种类似， 那些在肠道和口腔中的。我们的实验室证明，乳链菌肽是一种有效的抗微生物对关键 低浓度的牙周病细菌，包括Td，有可能破坏口腔生物膜的形成 在体外（11）。重要的是，尽管大多数乳链菌肽相关文献记载了其对革兰氏阳性菌的作用， 细菌，我们发表的工作（11）强调了其对革兰氏阴性菌的功效。此外，乳链菌肽在 低浓度也更有效地对抗某些口腔病原体对口腔细菌;使其 一种用于口服应用的潜在有用的和选择性的试剂。适当清除Td可提供更多 共生微生物组和“重置”稳态条件所需的修复，重塑或再生的 牙周组织因此，本文的主要假设是T.齿垢与PDL的相互作用 细胞介导对体内平衡和细胞功能的不利影响，导致受损的细胞 表型。此外，乳链菌肽是消除这些作用的潜在治疗剂。这一假设将 具体目的如下：1）确定牙本质素诱导的前体细胞活化的机制， 2）观察T.齿垢菌（Td）对PDL细胞过程的攻击， 3）确定亚抗微生物剂量的乳链菌肽是否可以消除Td/牙本质素介导的分化; MMP-2在PDL细胞中的活化。这一提议的成功将提供分子证据， TD介导的不良细胞病变效应导致严重牙周炎的特征;以及证据 乳酸链球菌素作为一种潜在的治疗药物，可在体外减少或逆转TD介导的牙周病变过程。

Project Summary/Abstract Periodontal disease is characterized by destruction of the hard and soft tissues that comprise the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), which is mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Furthermore, pathogenic microbes are a catalyst for periodontal disease pathogenesis since the development of a dysbiosis of the microbiome enhances a persistent immune response. These bacterial pathogens interact with host tissue and thereby trigger adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. One implicated bacteria, the oral spirochete, Treponema denticola (T. denticola or Td), is highly associated with periodontal disease (5). We’ve demonstrated that a Td outer membrane protein complex called dentilisin, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells (26,27). Dentilisin exposure also triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, there is no mechanism known for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease, or potential treatment. One possible therapeutic to combat these interactions is nisin. Nisin is a naturally occurring bacteriocin, widely used food preservative, and probiotic agent naturally made by bacterial species similar to those in the gut and oral cavity. Our lab demonstrated that nisin is an effective anti-microbial against key periodontopathic bacteria at low concentrations, including Td, with the potential to disrupt oral biofilm formation in vitro (11). Importantly, although most nisin related literature documents its role against gram-positive bacteria, our published work (11) highlights its efficacy against gram-negative bacteria. Furthermore, nisin at low concentrations is also more effective against certain oral pathogenic versus commensal bacteria; making it a potentially useful and selective agent for oral applications. Proper clearance of Td may provide a more symbiotic microbiome and “reset” homeostatic conditions needed for repair, remodeling or regeneration of the periodontium. Thus, the main hypotheses of this proposal are that T. denticola interactions with PDL cells mediate adverse effects on homeostasis and cellular functions leading to a compromised cellular phenotype. Additionally, nisin is a potential therapeutic for abrogating these effects. This hypothesis will be tested in the following specific aims: 1) Determine the mechanism of dentilisin-induced activation of pro- MMP-2 in PDL cells; 2) Characterize the effects of T. denticola(Td) challenge on PDL cell processes and differentiation; 3) Determine whether sub-antimicrobial doses of nisin can abrogate Td/dentilisin-mediated activation of MMP-2 in PDL cells. Success of this proposal will provide molecular evidence for a mechanism of Td-mediated adverse cytopathic effects leading to characteristics of severe periodontitis; as well as evidence on nisin as a potential therapeutic for reducing or reversing Td-mediated periodontopathic processes in vitro.