Study of Actinbacillus Actinomycetemcomitans Virulence

描述（由申请人提供）：牙周病原体，聚集杆菌（原放线菌）放线菌，表达几种复杂的多基因细胞毒素系统，对人类牙周组织健康重要的特定类型的细胞产生负面影响。这些系统包括白质毒素和细胞致死膨胀毒素（Cdt）的基因位点。这种令人印象深刻的毒力因子在其他口腔细菌病原体中尚未发现。我们之前的研究表明，从健康牙周状态转化为患病牙周状态的受试者中分离的放线菌菌株产生Cdt，导致DNA损伤并阻止原代人牙龈上皮细胞（HGEC）的细胞周期进展。该Cdt是由三个异源基因产物组成的非典型AB型毒素。本应用程序的目的是了解Cdt亚基的结构/功能关系，并定义亚基与原发性分化和未分化HGEC的具体相互作用。我们研究的一般假设是，每个亚基特有的功能域有助于HGEC的敏感性和靶向特异性。CdtA的主要作用是识别并结合毒素的细胞表面受体，而CdtC的主要作用是稳定CdtB在异源三聚体复合物中的结合并促进该亚基的细胞内运输。在定植和发病机制的背景下，排列在牙龈上的保护性上皮细胞层是Cdt的主要目标。上皮层是感染的物理屏障，并作为信号网络的一部分，提醒炎症细胞对微生物的攻击。Cdt优先抑制快速增殖的未分化和分化的牙龈上皮细胞，影响该层的生长和更新。对这一层的破坏将使放线菌和其他机会性细菌物种以及/或它们的产物更容易到达并攻击潜在的结缔组织和浸润性炎症细胞。研究的具体目的是：(1)鉴定和表征异毒素组装和与易感宿主细胞结合所需的CdtA和CdtC亚基的特定结构域；(2)评估CdtC亚基在CdtB组装、细胞内运输和细胞毒性中的作用；(3)利用cdt亚基突变体和中国仓鼠卵巢（CHO）细胞突变体获得体内特定亚基功能和相互作用的详细信息；(4)建立并开始表征人类原代牙龈上皮细胞（HGEC）模型，以评估cdt的作用。我们期望这种方法将推进我们正在进行的Cdt结构/功能研究，并深入了解Cdt如何参与导致牙周病变发展的复杂级联事件，作为多微生物病因学的一部分。这一信息可用于治疗，以阻断Cdt活性，以减少组织破坏的严重程度，这是牙周病的标志。

DESCRIPTION (provided by applicant): The periodontal pathogen, Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, expresses several complex multi-gene cytotoxin systems that negatively impact specific types of cells important to the health of the human periodontium. These systems include genetic loci for a leukotoxin and a cytolethal distending toxin (Cdt). This impressive repertoire of virulence factors has not been found in other oral bacterial pathogens. Our prior studies demonstrated that strains of A. actinomycetemcomitans, isolated from subjects that have converted from a healthy to diseased periodontal state, produce a Cdt that causes DNA damage and arrest of cell cycle progression of primary human gingival epithelial cells (HGEC). This Cdt is an atypical AB type toxin composed of three heterologous gene products. The objectives of this application are to understand the structure/function relationships of the Cdt subunits and to define the specific interactions of the subunits with primary differentiated and undifferentiated HGEC. The general hypothesis of our study is that functional domains unique to each subunit contribute to the exquisite sensitivity and target specificity of HGEC. The primary role of CdtA is to recognize and bind to the cell surface receptor for the toxin while that of CdtC is to stabilize CdtB binding in the heterotrimer complex and to facilitate the intracellular transport of this subunit. In the context of colonization and pathogenesis the protective epithelial cell layer that lines the gingiva is the principal target for the Cdt. The epithelial layer is a physical barrier to infection and functions as part of a signaling network that alerts inflammatory cells to microbial assault. The Cdt preferentially inhibits the rapidly proliferating undifferentiated versus differentiated gingival epithelial cells affecting the growth and turnover of this layer. Damage to this layer would allow A. actinomycetemcomitans as well as other opportunistic bacterial species, and/or their products, to more easily reach and attack the underlying connective tissue and infiltrating inflammatory cells. The specific aims of the study are: (1) to identify and characterize specific domains in the CdtA and CdtC subunits required for heterotoxin assembly and binding to susceptible host cells, (2) to assess the contributions of the CdtC subunit in the assembly, intracellular transport and cytotoxicity of CdtB, (3) to use the cdt subunit mutants and Chinese hamster ovary (CHO) cell mutants to obtain detailed information about specific subunit functions and interactions in vivo and (4) to develop and begin to characterize a primary human gingival epithelial cell (HGEC) model to assess the effects of the Cdt. We expect that this approach will advance our ongoing structure/function studies of the Cdt and provide insight into how the Cdt may contribute to the complex cascade of events that lead to the development of periodontal lesions as part of a polymicrobial etiology. This information can be exploited therapeutically to block Cdt activity to reduce the severity of the tissue destruction that is a hallmark of periodontal disease. PUBLIC HEALTH RELEVANCE: The oral bacterial pathogen Aggregatibacter actinomycetemcomitans produces a cytotoxin, known as the cytolethal distending toxin. This toxin causes DNA damage that signals the inhibition of growth of human gingival epithelial cells. Disruption of the protective epithelial cell layer formed in the gingival tissues gives opportunistic bacteria and their products access to the underlying connective tissue and infiltrating inflammatory cells important for the maintenance of a healthy periodontium.