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Abstract：
Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).
摘要：
牙周病的特征在于包含牙周组织的硬组织和软组织的破坏。这种破坏转化为细胞外基质（ECM）的降解，由细菌蛋白酶介导，宿主来源的基质金属蛋白酶（MMPs），以及宿主组织和免疫细胞释放的其他蛋白酶。细菌病原体与宿主组织相互作用，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。口腔螺旋体与牙周病高度相关。牙本质素,一种Denticola外膜蛋白复合物，有助于牙周膜（PDL）细胞中pro-MMP-2的慢性激活，并引发PDL细胞中活性MMP-2的激活剂和效应子的表达水平增加。尽管取得了这些进展,还不知道牙本质素诱导的MMP-2激活或PDL细胞病变行为导致疾病的机制。这里,我们描述了一种从T.denticola中纯化大量牙本质蛋白酶复合物的方法，并证明了其激活MMP-2的能力，MMP-2是牙周组织稳态的关键调节剂。本文提出的T.denticoladentilisin和MMP-2激活模型可能为dentilisin蛋白提供新的见解，并为进一步研究确定潜在的治疗靶标。关键特征•该协议建立在Cunningham等人描述的方法上。[1]用于螺旋体外膜蛋白的选择性释放。•我们调整了生物活性纯化的协议，去污剂稳定的外膜蛋白复合物来自T.denticola的大批量培养。•该方案涉及使用491型制备细胞的大规模制备性电泳。•然后我们使用明胶酶谱通过其激活基质金属蛋白酶2（MMP-2）的能力来证明纯化的牙本质蛋白复合物的活性。
