Study of Actinbacillus Actinomycetemcomitans Virulence

描述（由申请人提供）：牙周病原体，伴放线放线杆菌（Aggregatibacter）（原放线杆菌），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。这些系统包括白细胞毒素和细胞致死膨胀毒素（Cdt）的遗传基因座。这种令人印象深刻的毒力因子库尚未在其他口腔细菌病原体中发现。我们的前期研究表明，A.分离自从健康牙周状态转变为患病牙周状态的受试者的伴放线菌产生Cdt，其引起DNA损伤并阻止原代人牙龈上皮细胞（HGEC）的细胞周期进程。该Cdt是由三种异源基因产物组成的非典型AB型毒素。本申请的目的是了解Cdt亚基的结构/功能关系，并定义亚基与初级分化和未分化的HGEC的特异性相互作用。我们研究的一般假设是，每个亚基独特的功能结构域有助于HGEC的灵敏度和靶特异性。CdtA的主要作用是识别并结合毒素的细胞表面受体，而CdtC的主要作用是稳定异源三聚体复合物中的CdtB结合并促进该亚基的细胞内转运。在定殖和发病机制的背景下，衬在牙龈上的保护性上皮细胞层是Cdt的主要靶点。上皮层是感染的物理屏障，并作为信号网络的一部分，提醒炎症细胞微生物攻击。Cdt优先抑制快速增殖的未分化牙龈上皮细胞与分化牙龈上皮细胞，影响该层的生长和周转。这一层的损坏将使A.伴随放线菌以及其它机会性细菌物种和/或它们的产物，以更容易地到达和攻击下面的结缔组织和浸润的炎性细胞。这项研究的具体目标是：（1）鉴定和表征异源毒素组装和与易感宿主细胞结合所需的CdtA和CdtC亚基中的特异性结构域，（2）评估CdtC亚基在CdtB的组装、细胞内转运和细胞毒性中的作用，（3）使用cdt亚基突变体和中国仓鼠卵巢（CHO）细胞突变体来获得关于特定亚基功能和体内相互作用的详细信息，以及（4）开发并开始表征原代人牙龈上皮细胞（HGEC）模型以评估Cdt的作用。我们希望这种方法将推进我们正在进行的结构/功能的Cdt的研究，并提供深入了解如何Cdt可能有助于复杂的级联事件，导致牙周病变的发展作为一个多微生物病因的一部分。这些信息可以在治疗上用于阻断Cdt活性，以降低作为牙周病标志的组织破坏的严重程度。 公共卫生相关性：口腔细菌病原体伴放线聚集杆菌产生一种细胞毒素，称为细胞致死膨胀毒素。这种毒素导致DNA损伤，这是抑制人类牙龈上皮细胞生长的信号。牙龈组织中形成的保护性上皮细胞层的破坏使机会性细菌及其产物进入下层结缔组织和浸润性炎性细胞，这对于维持健康的牙周组织是重要的。

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.