Role of cytolethal distending toxin in periodontitis

描述(申请人提供)：作为人类最常见的细菌性疾病之一，牙周病困扰着多达50%的成年人，并导致支撑牙齿的结缔组织和骨骼的破坏。放线杆菌(Aggregatibacter，Aggregatibacter，放线杆菌)伴生放线菌(Aggregatibacter，AA)是一种与多种形式的人类牙周疾病相关的口腔细菌。有趣的是，AA产生的毒力因子似乎针对的是宿主免疫细胞，而不是支持牙齿的组织。我们已经在AA中发现了一种与大肠杆菌细胞致死性膨胀毒素(CDT)有关的毒素，但不同的是它针对的是淋巴细胞而不是上皮细胞。我们的假设是，AA CDT导致免疫抑制，从而允许AA成功地大量定植于人牙龈。然而，CDT在人类牙周病发病机制中的具体作用仍有待确定。我们的初步数据和与杜氏杆菌密切相关的CDT在巨噬细胞和树突状细胞上的数据表明，这种毒素诱导了非增殖性免疫细胞的细胞死亡。这些数据表明，细胞周期进程不是细胞毒性所必需的，CDT的作用机制可能比之前认为的更复杂。本研究的目的是了解CDT是否通过靶向非增殖性免疫细胞而促进牙周病，并确定该毒素是否代表了干预人类牙周病的潜在治疗靶点。在我们的第一个目标中，我们将确定CDT中毒的机制是否与非增殖性免疫细胞和上皮细胞相似。这将通过测量非增殖性巨噬细胞、树突状细胞和淋巴细胞相对于增殖性淋巴细胞和上皮细胞的致死性、结合、内化和DMA损伤来实现。在我们的第二个目标中，我们将产生一个不能表达CDT的AA突变体，以便使用牙周炎的小鼠模型来评估这种微生物的毒力。小鼠将被口服感染正常或CDT缺陷的AA生物，并将测量支持牙齿的牙槽骨丢失的程度、细菌数量和渗透的免疫细胞的生存能力。这种毒素在牙周病中的体内作用的成功证明将证实CDT在AA感染中的作用。了解CDT的生物学作用及其作用机制将使我们能够将其作为牙周病治疗干预的新的潜在靶点进行研究，从而为抗生素的使用提供一种替代方法。此外，抑制CDT引起的免疫抑制的化合物也可能对提高其他免疫抑制疾病的免疫功能有用。

DESCRIPTION (provided by applicant): As one of the most common bacterial diseases of humans, periodontal disease afflicts as much as 50% of the adult population and results in destruction of the connective tissues and bone that anchor the teeth. Aggregatibacter (Actinobacillus) actinomycetemcomitans (Aa) is an oral bacterium that is associated with various forms of human periodontal diseases. Interestingly, the factors produced by Aa that are involved in virulence appear to target host immune cells rather than the tissues that support the teeth. We have identified a toxin in Aa that is related to the E. coli cytolethal distending toxin (Cdt), but differs by targeting lymphocytes rather than epithelial cells. Our hypothesis is that the Aa Cdt causes immunosuppression, thus allowing Aa to successfully colonize the human gingiva in high numbers. However, the specific role that Cdt plays in the pathogenesis of human periodontal disease remains to be determined. Our preliminary data and data from the closely related H. ducreyi Cdt on macrophages and dendritic cells suggest that the toxin induces cell death in non-proliferating immune cells. These data suggest that cell cycle progression is not required for cytotoxicity and that the mechanism of Cdt action may be more complicated than previously thought. The goal of this study is to understand if Cdt contributes to periodontal disease by targeting non- proliferating immune cells, and to determine if the toxin represents a potential therapeutic target for intervention against human periodontal diseases. In our first Aim, we will determine if the mechanism of Cdt intoxication is similar for non-proliferating immune cells and epithelial cells. This will be accomplished by measuring lethality, binding, internalization, and DMA damage to non-proliferating macrophages, dendritic cells, and lymphocytes compared to proliferating lymphocytes and epithelial cells. In our second Aim, we will generate a mutant of Aa that is unable to express Cdt in order to assess the virulence of this organism using a mouse model of periodontitis. Mice will be infected orally with either normal or Cdt-defective Aa organisms and the degree of alveolar bone loss supporting the teeth, number of bacteria, and viability of infiltrating immune cells will be measured. The successful demonstration of an in vivo role for this toxin in periodontal disease will confirm a role for Cdt in Aa infections. Understanding the biological role of Cdt and its mechanism of action will allow us to investigate it as a new potential target for therapeutic intervention of periodontal disease, thus providing an alternative to the use of antibiotics. Furthermore, compounds that inhibit immunosuppression caused by Cdt may also have utility to increase immune function in other immunosuppressive diseases.