Study of Actinobacillus Actinomycetemcomitans Virulence

牙周病原体，Aggregatibacter（以前的Actinobacillus）actinomycetemcomitans， 几种复杂的多基因细胞毒素系统，其负面影响对免疫系统重要的特定类型的细胞。 人的牙周健康。这些系统包括白细胞毒素和致死细胞毒素的遗传基因座。 膨胀毒素（Cdt）。这种令人印象深刻的毒力因子库尚未在其他口腔细菌中发现。 病原体由细菌细胞毒素引起的破坏改变了局部环境， 微生物定植和宿主炎症。我们的前期研究表明，A. 从从健康牙周炎转化为患病牙周炎的受试者中分离的放线菌共生菌， 状态产生Cdt，其引起原代人口腔上皮细胞在G2/M间期的生长停滞 细胞周期。该Cdt是由三种基因产物组成的非典型AB型毒素。活性亚基， CdtB表现出DNA损伤活性，这使人联想到哺乳动物I型脱氧核糖核酸酶。CdtA， 可能与CdtC协同作用，将毒素结合到细胞表面。本申请的目的是 理解三个Cdt亚基的结构/功能关系，并定义特定的相互作用 原代分化和未分化的人牙龈上皮细胞（HGEC）的亚基。的 我们研究的一般假设是，三个Cdt亚基中的每一个都有特定的、独特的功能域， 有助于上皮细胞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的作用是稳定CdtB在细胞中的结合。 该亚基可以与异源三聚体复合物结合并促进该亚基的细胞内转运。在殖民化的背景下， 和发病机制，未分化的HGEC比分化的HGEC更可能是Cdt的靶点。 本申请包含以下具体目标：（1）鉴定和表征生物样品中的特定结构域。 CdtA和CdtC亚基是杂毒素组装和与易感宿主细胞结合所需的，（2）以评估 CdtC亚基在CdtB的组装、细胞内转运和细胞毒性中的贡献，（3）使用 cdt亚基突变体和中国仓鼠卵巢（CHO）细胞突变体，以获得有关 特异性亚基功能和体内相互作用，以及（4）发展并开始表征主要的 人牙龈上皮细胞（HGEC）模型，以评估Cdt的影响。我们希望我们的方法将 推进我们正在进行的Cdt结构/功能研究及其在细菌毒力中的可能作用， 提供了关于全毒素组装、HGEC中的毒性机制和细胞毒性的新的详细信息。 这些相互作用可以在治疗上用于阻断Cdt活性以降低组织损伤的严重性。 牙周病的症状有哪些？

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.