Study of Actinobacillus Actinomycetemcomitans Virulence

牙周病原体Aggregatibacter(前身为放线杆菌)伴生放线菌表达 几个复杂的多基因细胞毒素系统，对特定类型的细胞产生负面影响，对 人类牙周组织的健康。这些系统包括一种白毒素和一种细胞致死的遗传位点。 膨胀毒素(CDT)。这种令人印象深刻的毒力因子在其他口腔细菌中还没有发现 病原体。细菌细胞毒素造成的破坏改变了当地的环境，导致 微生物定植和宿主炎症。我们先前的研究表明，A. 从从健康牙周转化为疾病牙周的受试者中分离到的伴生放线菌 国家产生一种CDT，导致原代人类口腔上皮细胞生长停滞在G2/M间期 对细胞周期的影响。这种CDT是一种非典型的AB型毒素，由三个基因产物组成。活性亚基， CDtB，显示出一种DNA损伤活性，这让人想起哺乳动物的I型脱氧核糖核酸酶。CdtA， 可能与CdtC结合，将毒素结合到细胞表面。此应用程序的目标是 了解三个CDT亚基的结构/功能关系，并定义具体的相互作用 具有原代分化和未分化的人牙龈上皮细胞(HGEC)的亚基。这个 我们研究的一般假设是CDT三个亚基中每一个都有特定的、独特的功能域 有助于提高上皮细胞的敏感度和靶向性。CDtA的主要作用是 CDtC识别并结合到细胞表面的毒素受体，而CDtC的作用是稳定CDtB在细胞内的结合 异三聚体复合体和促进该亚单位的细胞内转运。在殖民背景下 在发病机制上，未分化的HGEC比分化的HGEC更有可能成为CDT的靶点。 本应用程序包含以下具体目标：(1)识别和描述 异源毒素组装和与敏感宿主细胞结合所需的CDtA和CDtC亚基，(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)使用 CDT亚单位突变体和中国仓鼠卵巢(CHO)细胞突变体获得详细信息 体内特定亚基的功能和相互作用以及(4)发展和开始表征初级 用人牙龈上皮细胞(HGEC)模型评价CDT的作用。我们预计，我们的方法将 推进我们正在进行的CDT的结构/功能研究及其在细菌毒力中的可能作用 提供全毒素组装、毒性机制(S)和细胞的新的详细信息 可用于治疗以阻断CDT活性以减轻组织严重程度的相互作用 破坏是牙周病的标志。

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. The destruction caused by bacterial cytotoxins alters the local environment leading to increased microbial colonization and host inflammation. 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 the growth arrest of primary human oral epithelial cells at the G2/M interphase of the cell cycle. This Cdt is an atypical AB type toxin composed of three gene products. The active subunit, CdtB, exhibits a DNA damaging activity that is reminiscent of mammalian type I deoxyribonucleases. CdtA, possibly in concert with CdtC, binds the toxin to the cell surface. The objectives of this application are to understand the structure/function relationships of the three Cdt subunits and to define the specific interactions of the subunits with primary differentiated and undifferentiated human gingival epithelial cells (HGEC). The general hypothesis of our study is that specific, unique functional domains in each of the three Cdt subunits contribute to the exquisite sensitivity and target specificity of epithelial cells. 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, undifferentiated HGEC present a more likely target for the Cdt than differentiated HGEC. This application contains the following specific aims: (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 our approach will advance our ongoing structure/function studies of the Cdt and its possible role in bacterial virulence by providing new detailed information about holotoxin assembly, mechanism(s) of toxicity in HGEC and cellular interactions that can be exploited therapeutically to block Cdt activity to reduce the severity of the tissue destruction that is a hallmark of periodontal disease.