Study of Actinobacillus Actinomycetemcomitans Virulence

描述：牙周病原体，伴生放线杆菌，表达几个复杂的多基因毒素系统，促进该细菌与人类细胞的负相互作用。这些系统包括入侵所需的基因和白毒素和细胞致死性扩张毒素(CDT)的遗传位点。这一令人印象深刻和广泛的潜在毒力因子谱系在其他口腔微生物病原体中尚未发现。本申请的重点是我们对伴生放线菌Y4的CDT研究的继续。这种毒素由三个基因产物组成，它们自组装形成一个三方复合体或全毒素。全息毒素的活性亚基是一种核酸酶，在功能上与哺乳动物的DNase I有关。DNase I样蛋白针对大多数真核细胞的细胞核，在那里它会导致广泛的DNA片段化和染色质损伤。这些活动导致在生长周期的G2阶段结束时生长停滞。这项应用的主要目的是了解CDT的不同亚基如何作用于产生生物活性毒素，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。其具体目的是：(1)确定CDT基因产物的相互关系；(2)确定CDT对培养的口腔上皮细胞和成纤维细胞的不同作用；(3)利用CHO细胞突变体，检测和表征CDT基因产物在中毒不同阶段的特定作用。天然组装的重组全毒素和从纯化的重组CDT蛋白体外组装的全毒素将用于细胞毒性和结合实验，以检测亚基的功能。CDT对新近永生化的人口腔上皮细胞系和人牙周膜成纤维细胞的不同作用将被检测，以确定CDT的特异性和宿主范围。在一种新的方法中，对各种CDT亚基的活性具有抗性的CHO细胞突变体将被分离并用于分离亚基的活性以供研究。我们研究的长期目标是深入了解伴放线放线杆菌CDT在扰乱正常细胞功能中的作用，这对维持人类口腔的健康状态很重要。

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.