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

项目摘要/摘要 牙周病的特征是组成牙周的硬组织和软组织遭到破坏。 牙周组织。这种破坏转化为细胞外基质(ECM)的降解，这是 由细菌蛋白酶、宿主衍生的基质金属蛋白酶(MMPs)和其他蛋白酶介导 由宿主组织和免疫细胞释放。此外，病原微生物也是牙周的催化剂。 疾病的发病机制由于微生物群的失调而增强了持久的免疫力 回应。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。一种牵连的细菌， 口腔螺旋体，齿密螺旋体(T.denticola或TD)，与牙周病高度相关(5)。 我们已经证明了一种名为牙本质蛋白的TD外膜蛋白复合体，有助于慢性 牙周膜(PDL)细胞(26，27)中原-基质金属蛋白酶-2的激活。接触牙菌素也会引发增加 活化的基质金属蛋白酶-2激活物和效应物在PDL细胞中的表达水平。尽管取得了这些进步，但没有 已知的机制是牙菌素诱导的基质金属蛋白酶-2激活或PDL细胞病变行为导致疾病，或 潜在的治疗方法。对抗这些相互作用的一种可能的治疗方法是Nisin。Nisin是一种自然产生的 细菌素，广泛使用的食品防腐剂和益生菌剂，由类似于 肠道和口腔中的细菌。我们的实验室证明Nisin是一种有效的抗菌素。 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 体外培养(11)。重要的是，尽管大多数与Nisin相关的文献记载了它在对抗革兰氏阳性菌中的作用 细菌，我们发表的工作(11)强调了它对革兰氏阴性细菌的有效性。此外，乳酸链球菌素在 低浓度对某些口腔致病菌也比共生菌更有效； 一种潜在的有用和选择性的口腔用药。运输署的适当许可可能会提供更多 共生微生物群和修复、重塑或再生所需的动态平衡条件 牙周组织。因此，这一建议的主要假设是齿状毛滴虫与PDL相互作用。 细胞调节动态平衡和细胞功能的不利影响，导致细胞受损 表型。此外，Nisin是一种潜在的消除这些影响的治疗方法。这一假说将 试验的具体目的如下：1)确定牙菌素诱导前体细胞活化的机制。 2)研究齿状毛滴虫(Td)攻击对PDL细胞突起和细胞周期的影响。 3)确定亚抗菌剂量的乳链菌素是否能消除TD/Didlisin介导的 PDL细胞中基质金属蛋白酶-2的激活。这一提议的成功将为以下机制提供分子证据 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 Nisin作为一种潜在的治疗方法在体外减少或逆转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.