Study of Actinbacillus Actinomycetemcomitans Virulence

描述(由申请人提供)：牙周病原体Aggregatibacter(以前的放线杆菌)伴生放线菌表达几个复杂的多基因细胞毒素系统，对对人类牙周组织健康至关重要的特定类型的细胞产生负面影响。这些系统包括一种白毒素和一种细胞致死性膨胀毒素(CDT)的遗传位点。这一令人印象深刻的毒力因子谱系在其他口腔细菌病原体中尚未发现。我们先前的研究表明，从健康牙周状态转变为疾病牙周状态的受试者中分离的伴放线放线菌菌株，会产生一种CDT，导致原代人类牙龈上皮细胞(HGEC)DNA损伤和细胞周期停滞。该CDT是一种由三种异源基因产物组成的非典型AB型毒素。本应用的目的是了解CDT亚基的结构/功能关系，并确定亚基与原发、分化和未分化的HGEC的具体相互作用。我们研究的一般假设是，每个亚单位独特的功能域有助于HGEC的精致敏感性和靶向性。CDtA的主要作用是识别和结合毒素的细胞表面受体，而CDtC的主要作用是稳定异三聚体中的CDtB结合，并促进该亚单位的细胞内转运。在定植和发病机制的背景下，位于牙龈内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，是信号网络的一部分，向炎症细胞发出微生物攻击的警报。CDT优先抑制快速增殖的未分化与分化的牙龈上皮细胞，影响这一层的生长和周转。对这一层的破坏将使伴生放线菌以及其他机会性细菌物种和/或其产物更容易到达和攻击潜在的结缔组织和浸润性炎症细胞。这项研究的具体目的是：(1)确定和表征异源毒素组装和与敏感宿主细胞结合所需的特定结构域；(2)评估CDtC亚基在CDtB组装、细胞内转运和细胞毒性中的作用；(3)利用CDT亚单位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；以及(4)建立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CDT的结构/功能研究，并提供对CDT如何在导致牙周病变的复杂级联事件中的作用的洞察，这些事件是多菌病原学的一部分。这一信息可用于治疗，以阻断CDT活性，以减轻牙周病的标志组织破坏的严重程度。 与公共卫生相关：口腔细菌病原体Aggregatibacter放线菌伴生菌会产生一种细胞毒素，称为细胞致死性膨胀毒素。这种毒素会导致DNA损伤，从而抑制人类牙龈上皮细胞的生长。牙龈组织中形成的保护性上皮细胞层的破坏使机会细菌及其产品能够进入底层结缔组织和渗透的炎症细胞，这对维持健康的牙周组织非常重要。

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.