Study of Actinobacillus Actinomycetemcomitans Virulence

伴放线放线杆菌毒力研究

• 批准号: 6688006
• 负责人: JOSEPH M. DIRIENZO
• 金额: $ 31.7万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6688006
• 关键词: Actinobacillus actinomycetemcomitans; CHO cells; SDS polyacrylamide gel electrophoresis; bacterial cytopathogenic effect; bacterial genetics; bacterial toxicology; bacterial toxins; cytotoxicity; epithelium; fibroblasts; flow cytometry; human tissue; immunoprecipitation; mutant; oral bacteria; site directed mutagenesis; tissue /cell culture; virulence; western blottings

DESCRIPTION: The periodontal pathogen, Actinobacillus actinomycetemcomitans, expresses several complex multi-gene toxin systems that promote the negative interaction of this bacterium with human cells. These systems include genes required for invasion and genetic loci for a leukotoxin and a cytolethal-distending toxin (CDT). This impressive and extensive repertoire of potential virulence factors has not been found in other oral microbial pathogens. The focus of this application is a continuation of our studies of the CDT of A. Actinomycetemcomitans Y4. This toxin is composed of three gene products which self-assemble to form a tripartite complex or holotoxin. The active subunit of the holotoxin is a nuclease, which is functionally related to mammalian DNase I. The DNase I-like protein targets the nucleus of most eukaryotic cells where it causes extensive DNA fragmentation and chromatin damage. These activities lead to growth arrest at the end of the G2 phase of the growth cycle. The major objectives of this application are to understand how the various subunits of the CDT function to produce a biologically active toxin, to define the interactions of the CDT with cell lines that are relevant to the integrity of the human oral cavity and to isolate and use CHO cell mutants to study cytotoxicity. The Specific Aims are: (i) to determine the interrelationships of the cdtA, cdtB and cdtC gene products, (ii) to determine the differential effects of the CDT on cultured oral epithelial cells and fibroblasts, and (iii) to use CHO cell mutants to examine and characterize specific roles of cdt gene products in the various stages of intoxication. Natively assembled recombinant holotoxin and holotoxin assembled in vitro, from purified recombinant Cdt proteins, will be used in cytotoxicity and binding experiments to examine the functions of the subunits. The differential effects of the CDT on a recently immortalized human oral epithelial cell line and human periodontal ligament fibroblasts will be examined to define the specificity and host range of the CDT. In a novel approach, CHO cell mutants resistant to the activities of the various CDT subunits will be isolated and used to segregate the subunit activities for study. The long-term goal of our study is to gain insight into the role of the Actinobacillus actinomycetemcomitans CDT in the perturbation of normal cell functions that are important for maintaining the healthy status of the human oral cavity.

产品说明：牙周病病原体，放线共生放线杆菌，表达几个复杂的多基因毒素系统，促进这种细菌与人类细胞的负面相互作用。这些系统包括入侵所需的基因和白细胞毒素和细胞致死膨胀毒素（CDT）的遗传位点。这种令人印象深刻和广泛的潜在毒力因子库尚未在其他口腔微生物病原体中发现。本申请的重点是继续我们的研究的CDT A。伴放线菌Y4.这种毒素由三种基因产物组成，它们自组装形成三重复合物或全毒素。全毒素的活性亚基是一种核酸酶，其功能与哺乳动物DNA酶I相关。DNA酶I样蛋白靶向大多数真核细胞的细胞核，在那里它引起广泛的DNA片段化和染色质损伤。这些活动导致生长周期G2期结束时的生长停滞。本申请的主要目的是了解CDT的各种亚基如何发挥作用以产生生物活性毒素，以确定CDT与与人类口腔完整性相关的细胞系的相互作用，并分离和使用CHO细胞突变体来研究细胞毒性。具体目标是：（i）确定cdtA、cdtB和cdtC基因产物的相互关系，（ii）确定CDT对培养的口腔上皮细胞和成纤维细胞的不同作用，和（iii）使用CHO细胞突变体来检查和表征在中毒的各个阶段中CDT基因产物的特定作用。天然组装的重组全毒素和体外组装的全毒素，从纯化的重组Cdt蛋白，将用于细胞毒性和结合实验，以检查亚基的功能。将检查CDT对最近永生化的人口腔上皮细胞系和人牙周膜成纤维细胞的差异效应，以确定CDT的特异性和宿主范围。在一种新的方法中，将分离对各种CDT亚基的活性具有抗性的CHO细胞突变体，并将其用于分离亚基活性以供研究。我们研究的长期目标是深入了解伴放线放线杆菌CDT在干扰正常细胞功能中的作用，这些功能对维持人类口腔的健康状态至关重要。