REGULATION OF LEUKOTOXIN IN A ACTINOMYCETEMCOMITANS

放线菌共生体中白细胞毒素的调节

• 批准号: 2770261
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 23.39万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770261
• 关键词: Actinobacillus actinomycetemcomitans; DNA binding protein; bacterial RNA; bacterial genetics; bacterial toxins; gene expression; gene mutation; genetic library; genetic mapping; genetic promoter element; genetic regulation; genetic regulatory element; genetic strain; genetic transcription; mutant; nucleic acid sequence; plasmids; polymerase chain reaction; protein biosynthesis; reporter genes; transcription factor; virulence

Periodontitis, an inflammatory disease of tissues in the subgingival crevice, is associated with a dramatic shift in the subgingival microflora towards predominantly gram negative organisms. To understand why certain bacteria are successful pathogens, it is important to determine the structure, function, and regulation of each of their virulence factors. Actinobacillus actinomycetemcomitans has been strongly implicated in Localized Juvenile Periodontitis and in several adult periodontal disorders. The primary candidate virulence factor produced by A. actinomycetemcomitans is a leukotoxin which kills host neutrophils. Bacterial isolates associated with pathogenesis usually produce high levels of leukotoxin. Moreover, the levels of this virulence factor are regulated by changes in the environment; both anaerobic and high salt conditions favor increased production of leukotoxin. We now propose to exploit the molecular genetic tools we have developed in order to determine the mechanisms of strain-specific and environmental regulation of the leukotoxin. Both genetic (transposon mutagenesis, complementation and gene-deletion) and biochemical (gel mobility shift, affinity chromatography) approaches will be used to identify the leukotoxin promoter sequences critical in regulation and to characterize the proteins that are involved. The genes encoding these regulatory proteins will be cloned and used to generate specific mutations in the endogenous A. actinomycetemcomitans genes. The resulting mutants, deficient in various leukotoxin regulatory pathways, will be tested for defects in general and in environmental regulation. Importantly, these mutant strains of A. actinomycetemcomitans can be used in animal models in future studies to assess the role of individual regulatory pathways in the pathogenesis of A. actinomycetemcomitans. Knowledge of the mechanisms of regulation of virulence factors may lead to the future design of novel drugs that target transcription factors involved in regulating several different virulence components.

牙周炎是一种龈下组织的炎症性疾病 与龈下微生物区系的急剧变化有关 主要是革兰氏阴性菌。为了理解为什么某些 细菌是成功的病原体，重要的是要确定 结构，功能和调节它们的每一个毒力因子。 伴放线放线杆菌与 局限性青少年牙周炎和几种成人牙周炎 紊乱 A. 伴放线菌是一种杀死宿主中性粒细胞的白细胞毒素。 与发病机制相关的细菌分离株通常产生高 白细胞毒素水平 此外，这种毒力因子的水平是 受环境变化的调节;厌氧和高盐 条件有利于增加白细胞毒素的产生。 我们现建议 利用我们开发的分子遗传工具， 确定菌株特异性和环境调节的机制 白细胞毒素 两种遗传（转座子诱变，互补 和基因缺失）和生物化学（凝胶迁移率变化，亲和力 色谱法）的方法将用于鉴定白细胞毒素 启动子序列在调控中起关键作用， 有牵连的人编码这些调节蛋白的基因将被 克隆并用于在内源性A. actinomycetemcomitans基因由此产生的突变体，缺乏各种 白细胞毒素调节途径，将测试一般缺陷， 在环境监管方面。重要的是，这些A. 在未来的研究中，放线菌共生菌可用于动物模型， 评估个体调节途径在糖尿病发病机制中的作用 A.伴放线菌对调节机制的了解 毒力因子可能会导致未来设计新的药物， 参与调节几种不同毒力的转录因子 件.