REGULATION OF LEUKOTOXIN IN A ACTINOMYCETEMCOMITANS

牙周炎，一种牙龈下组织的炎症性疾病 缝隙，与龈下微生物区系的戏剧性变化有关 以革兰氏阴性为主的微生物。要了解为什么某些 细菌是成功的病原体，重要的是要确定 它们各自毒力因子的结构、功能和调控。 伴生放线杆菌被强烈地牵连到 局限性青少年牙周炎和几种成人牙周炎 精神错乱。产生的主要候选毒力因子。 伴生放线菌是一种杀死宿主中性粒细胞的白毒素。 与致病相关的细菌分离株通常产生高水平的 白血球毒素水平。此外，这种毒力因子的水平是 受环境变化调节的；厌氧的和高盐的 条件有利于增加白毒素的产生。我们现在建议 利用我们开发的分子遗传学工具 确定菌株特异性和环境调节的机制 白毒素的含量。既有遗传(转座子突变、互补 和基因缺失)和生化(凝胶迁移率变化、亲和力 层析)方法将被用来鉴定白毒素 启动子序列在调控和鉴定蛋白质中起关键作用 都牵涉其中。编码这些调节蛋白的基因将是 克隆并用于在内源A. 伴生放线菌基因。由此产生的突变体，缺乏不同的 白毒素调节途径，将测试一般缺陷和 在环境监管方面。重要的是，这些突变的A. 放线菌伴生菌可在未来的研究中用于动物模型 评估个体调控通路在急性髓细胞白血病发病机制中的作用 伴生放线菌。对生物多样性调节机制的认识 毒力因素可能导致未来针对以下目标的新药设计 参与调节几种不同毒力的转录因子 组件。

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.