Study of Actinbacillus Actinomycetemcomitans Virulence

伴放线放线杆菌毒力研究

• 批准号: 8299179
• 负责人: JOSEPH M. DIRIENZO
• 金额: $ 38.42万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299179
• 关键词: Actinobacillus actinomycetemcomitans; Adult; Affect; Amino Acid Substitution; Animal Model; Appearance; Bacteria; Binding; Biological; Biological Models; Bulla; Cell Cycle Arrest; Cell Cycle Progression; Cell Surface Receptors; Cell model; Cells; Characteristics; Chinese Hamster Ovary Cell; Coculture Techniques; Complex; Connective Tissue; Cytotoxin; DNA Damage; Development; Disease; Epithelial; Epithelial Cells; Etiology; Event; Exhibits; Genes; Genetic; Gingiva; Gram-Negative Bacteria; Grant; Growth; Health; Horizontal Gene Transfer; Human; Individual; Infection; Inflammatory; Inflammatory Infiltrate; Intracellular Transport; Invaded; Juvenile Periodontitis; Kinetics; Lead; Lesion; Maintenance; Membrane; Mutagens; Oral; Oral cavity; Pathogenesis; Periodontal Diseases; Periodontitis; Periodontium; Porphyromonas gingivalis; Proliferating; Property; Proteins; Role; Series; Severities; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Structure-Activity Relationship; Surface; System; Tissues; Toxin; Undifferentiated; Virulence; Virulence Factors; assault; base; cell type; cytolethal distending toxin; cytotoxicity; extracellular; human disease; in vivo; insight; leukotoxin; microbial; mutant; oral pathogen; pathogen; public health relevance; research study

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.

描述（由申请人提供）：牙周病原体 Aggregatibacter（以前称为 Actinobacillus）actinomycetemcomitans，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）的遗传位点。在其他口腔细菌病原体中尚未发现这种令人印象深刻的毒力因子。我们之前的研究表明，从健康牙周状态转变为患病牙周状态的受试者中分离出的 A. actinomycetemcomitans 菌株会产生 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是一种非典型AB型毒素，由三个异源基因产物组成。该应用的目的是了解 Cdt 亚基的结构/功能关系，并定义亚基与初级分化和未分化 HGEC 的特定相互作用。我们研究的总体假设是每个亚基独特的功能域有助于 HGEC 的精确敏感性和靶点特异性。 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。在定植和发病机制的背景下，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，是信号网络的一部分，提醒炎症细胞受到微生物攻击。 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相比），从而影响该层的生长和周转。该层的损坏将使伴放线放线菌以及其他机会性细菌种类和/或其产物更容易到达并攻击下面的结缔组织和浸润炎症细胞。该研究的具体目的是：（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亚基中的特定结构域，（2）评估CdtC亚基在CdtB组装、细胞内运输和细胞毒性中的贡献，（3）利用cdt亚基突变体和中国仓鼠卵巢（CHO）细胞突变体获得 有关体内特定亚基功能和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 的效果。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 如何促成复杂的级联事件，从而导致作为多微生物病因学一部分的牙周病变的发展。该信息可用于治疗性阻断 Cdt 活性，从而减轻牙周病标志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性细菌及其产物能够进入下面的结缔组织和浸润炎症细胞，这对于维持健康的牙周组织很重要。