REGULATION OF LEUKOTOXIN IN A ACTINOMYCETEMCOMITANS

放线菌共生体中白细胞毒素的调节

• 批准号: 2131639
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 13.52万
• 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 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2131639
• 关键词: Actinobacillus actinomycetemcomitans; SDS polyacrylamide gel electrophoresis; acidity /alkalinity; bacterial genetics; bacterial proteins; bacterial toxins; beta galactosidase; enzyme activity; gene expression; gene mutation; genetic promoter element; genetic regulation; genetic transcription; gingiva; iron; microorganism culture; molecular cloning; northern blottings; nucleic acid sequence; periodontitis; protein biosynthesis; transcription factor; virulence; western blottings

Periodontitis, an inflammatory disease of tissues in the subgingival crevice, can lead to soft tissue damage and dramatic bone loss. Development of periodontitis is associated with a dramatic shift in the subgingival microflora; gram negative microorganisms replace gram positive bacteria as the primary flora. In order to understand why certain bacteria are successful periodontopathogens, it is important to determine how their virulence determinants might be regulated by components of the complex and unique subgingival microenvironment. This proposal focuses on regulation in the gram negative, coccobacillus Actinobacillus actinomycetemcomitans (Aa) for several reasons: (1) Aa is the primary candidate pathogen in several forms of periodontitis, (2) Aa is the only periodontopathogen to produce a leukotoxin, and (3) the Aa leukotoxin gene has been cloned, so both molecular genetic and biochemical approaches can be used to study its regulation. Our hypothesis is that changes in environmental cues will alter the expression of Aa leukotoxin. Aa strains in which both leukotoxin and beta-galactosidase are transcribed from the leukotoxin promoter have been generated. This will allow leukotoxin promoter activity to be assessed using a simple, quantitative beta-galactosidase assay. These Aa strains will be grown in media with varying amounts of iron, in media at different pHs and in cultures which have been stressed (heat shocked). Beta-galactosidase activity will be assayed to determine those culture changes that modulate leukotoxin expression. then RNA and protein will be isolated from the same cultures and analyzed using specific DNA and antibody probes to determine the level at which leukotoxin regulation occurs. the regulation of cell envelope proteins will also be examined from the same cells. The results will show how the virulence factor, leukotoxin, is regulated by changes in these three environmental signals and will also identify other regulated proteins. the other regulated proteins are candidate virulence factors. Using the cloned leukotoxin gene as a starting point, molecular genetic approaches will then be used to determine the DNA sequences and regulatory proteins involved in regulating leukotoxin transcription in Aa. Deletion and point mutant analyses of the leukotoxin promoter will define important cis elements. Transposon mutagenesis will be used to identify genes which regulate the transcriptional response of the leukotoxin gene. The results from these studies will define the cast of characters used by Aa to regulate a virulence factor, leukotoxin. this will enable us, in future years, to delineate the molecular mechanisms by which environmental cues regulate leukotoxin and other virulence factors of Aa.

牙周炎是一种龈下组织的炎症性疾病 裂缝会导致软组织损伤和严重的骨质流失。 牙周炎的发展与牙周组织的急剧变化有关。 龈下菌群;革兰氏阴性菌取代革兰氏阴性菌 阳性菌为主要植物群。 为了理解为什么 某些细菌是成功的牙周病原体，重要的是 确定它们的毒力决定因素如何受到 复杂而独特的龈下微环境的组成部分。 这 提案的重点是革兰氏阴性杆菌、球杆菌 放线共生放线杆菌（Actinobacillus actinomycetemcomitans，Aa）的产生有以下几个原因：（1）Aa 是几种牙周炎的主要候选病原体，（2） AA是唯一能产生白细胞毒素的牙周病病原体， Aa白细胞毒素基因已被克隆， 生物化学方法可用于研究其调控。 我们的假设是，环境线索的变化会改变 Aa白细胞毒素的表达。 其中白细胞毒素和 β-半乳糖苷酶是从白细胞毒素启动子转录的， 生成的. 这将允许评估白细胞毒素启动子活性 使用简单的定量β-半乳糖苷酶测定。 这些Aa菌株 将在含有不同量铁的培养基中生长， 不同的pH值和已强调（热休克）的文化。 将测定β-半乳糖苷酶活性以确定这些培养物 调节白细胞毒素表达的变化。 那么RNA和蛋白质 从相同的培养物中分离并使用特定的DNA进行分析， 抗体探针来确定白细胞毒素调节的水平， 发生。 还将研究细胞包膜蛋白的调节 来自相同的细胞。 结果将显示毒力因子， 白细胞毒素受这三种环境信号的变化调节 并且还将鉴定其他受调节的蛋白质。 另一个是监管 蛋白质是候选毒力因子。 以克隆的白细胞毒素基因为出发点， 然后将使用方法来确定DNA序列， 参与调节白细胞毒素转录的调节蛋白 AA. 白细胞毒素启动子的缺失和点突变分析将 确定重要的独联体要素。 转座子诱变将用于 鉴定调节细胞转录反应的基因， 白细胞毒素基因 这些研究的结果将确定 Aa用来调节毒力因子白细胞毒素的特征。 这 将使我们能够在未来几年中，描绘分子机制， 环境因素通过这种机制调节白细胞毒素和其他毒力 的因素。