REGULATION OF LEUKOTOXIN IN A ACTINOMYCETEMCOMITANS

牙周炎，一种牙龈下组织的炎症性疾病 缝隙，会导致软组织损伤和严重的骨质流失。 牙周炎的发生与牙周病的急剧变化有关 龈下微生物区系；革兰氏阴性菌取代革兰氏 菌群以阳性菌为主。为了理解为什么 某些细菌是成功的牙周病原体，重要的是要 确定它们的毒力决定因素可能如何通过 复杂而独特的龈下微环境的组成部分。这 建议重点监管革兰氏阴性杆菌、球杆菌 伴生放线杆菌(Aa)有几个原因：(1)Aa 是几种牙周炎的主要候选病原体，(2) AA是唯一一种产生白毒素的牙周病原体，以及(3) AA型白毒素基因已被克隆，因此无论是分子遗传学还是 生化方法可以用来研究它的调控。 我们的假设是，环境线索的变化将改变 AA白毒素的表达。在AA菌株中，白毒素和 β-半乳糖苷酶是从白毒素启动子转录而来的 已生成。这将允许评估白毒素启动子的活性。 使用一种简单、定量的β-半乳糖苷酶检测。这些AA菌株 将在含铁量不同的介质中生长，在介质中 不同的PHS和在受到压力(热休克)的文化中。 将测定β-半乳糖苷酶活性以确定这些培养物 调节白毒素表达的变化。那么RNA和蛋白质就会 从相同的培养物中分离并使用特定的DNA和 抗体探针用于确定白毒素调节的水平 发生。对细胞包膜蛋白的调节也将被研究。 来自相同的细胞。结果将显示毒力因素是如何， 白毒素，受这三种环境信号的变化调节 还将识别其他受调控的蛋白质。另一个受监管的 蛋白质是候选毒力因子。 以克隆的白毒素基因为起点，分子遗传学 然后将使用方法来确定DNA序列和 参与白毒素转录调控的调节蛋白 AA.白毒素启动子的缺失和点突变分析 定义重要的配置项元素。转座子诱变将用于 识别调控细胞转录反应的基因 白毒素基因。这些研究的结果将定义 AA用来调节一种毒性因子--白毒素的特征。这 将使我们在未来几年能够描绘出分子机制 通过环境线索来调节白毒素和其他毒力 AA的影响因素。

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.