Anaerobic Regulatory Pathways in a Periodontopathogen

牙周病原菌的厌氧调节途径

• 批准号: 6826488
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 32.85万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6826488
• 关键词: Actinobacillus actinomycetemcomitans; anaerobic bacteria; bacterial genetics; bacterial toxins; gene expression; genetic regulation; gingiva; microarray technology; northern blottings; periodontitis; polymerase chain reaction; transcription factor; virulence

DESCRIPTION: Periodontitis, an inflammatory disease of tissues in the subgingival crevice, is associated with a dramatic shift in the subgingival microflora towards Gram negative organisms. We have focused on studying the virulence properties of one of these periodontal pathogens, the bacterium Actinobacillus actinomycetemcomitans (Aa). This bacterium has been strongly implicated in localized aggressive periodontitis and in several adult periodontal disorders. Aa is a facultative anaerobe that is capable of colonizing both an anaerobic microenvironment, like the diseased periodontal pocket, and an aerobic environment, like the blood stream. Thus, Aa has evolved virulence mechanisms that allow it to shift between these two environments. Using two-dimensional protein gels, we have shown that anaerobic growth induces the synthesis of dozens of Aa proteins, including leukotoxin (a presumed virulence protein that kills neutrophils). Numerous other proteins are repressed under anaerobic conditions but induced in the presence of oxygen. By generating defined mutations in An, we have shown that the aerobic/anaerobic regulation of most of these proteins is controlled by the Fnr or ArcAB pathways, as expected. Nevertheless, interestingly, the regulation of at least eight other proteins, including leukotoxin, was not controlled by Fnr or ArcAB. Since Aa does not encode significant homologues to any other bacterial oxygen regulatory proteins, we posit that a subset of Aa genes will be controlled by one (or more) transcriptional regulatory pathways which have not previously been described in Aa or in any other bacterial system. The full complement of Aa genes that are differentially synthesized in aerobic versus anaerobic growth will be identified using DNA microarrays (Specific Aim I). Importantly, we will also identify the set of oxygen-responsive genes, like leukotoxin, that are regulated by the previously unidentified, non-Fnr/non-ArcA pathway(s). In Specific Aim II, a systematic and comprehensive molecular genetic approach will be employed to identify this potentially novel regulatory pathway and the transcription factors involved. Finally, the interactions of our newly identified oxygen regulatory proteins with redox-regulated Aa promoters will be characterized (Specific Aim III). This will allow us to begin to develop the first molecular models for the mechanisms of non-Fnr/non-ArcAB aerobic/anaerobic regulation in a periodontal pathogen. The research proposed is highly significant because it will reveal the players in a new regulatory pathway regulating the adaptation of An, and possibly other periodontal pathogens, to the anaerobic subgingival microenvironment. Characterizing undefined, potentially new transcriptional regulatory pathways is important because the proteins involved may serve as targets for future drug development.

描述：牙周炎是龈下缝隙组织的炎症性疾病，与龈下微生物群向革兰氏阴性菌的急剧转变有关。我们重点研究牙周病原体之一——伴放线放线杆菌（Aa）的毒力特性。这种细菌与局部侵袭性牙周炎和多种成人牙周病密切相关。 Aa 是一种兼性厌氧菌，能够定植于厌氧微环境（如患病的牙周袋）和需氧环境（如血流）。因此，Aa 已经进化出毒力机制，使其能够在这两种环境之间转换。使用二维蛋白质凝胶，我们发现厌氧生长会诱导数十种 Aa 蛋白质的合成，其中包括白细胞毒素（一种假定的杀死中性粒细胞的毒力蛋白质）。许多其他蛋白质在厌氧条件下受到抑制，但在有氧条件下被诱导。通过在 An 中产生确定的突变，我们发现大多数这些蛋白质的有氧/无氧调节是由 Fnr 或 ArcAB 途径控制的，正如预期的那样。然而，有趣的是，至少八种其他蛋白质（包括白细胞毒素）的调节不受 Fnr 或 ArcAB 控制。由于 Aa 不编码与任何其他细菌氧调节蛋白显着的同源物，因此我们假设 Aa 基因的子集将受到一个（或多个）转录调节途径的控制，这些途径以前在 Aa 或任何其他细菌系统中都没有描述过。将使用 DNA 微阵列来鉴定在需氧和厌氧生长中差异合成的完整 Aa 基因（具体目标 I）。重要的是，我们还将鉴定一组氧响应基因，如白细胞毒素，它们受先前未鉴定的非 Fnr/非 ArcA 途径的调节。在Specific Aim II中，将采用系统且全面的分子遗传学方法来识别这种潜在的新颖调控途径和所涉及的转录因子。最后，我们将表征我们新鉴定的氧调节蛋白与氧化还原调节 Aa 启动子的相互作用（具体目标 III）。这将使我们能够开始开发第一个关于牙周病原体非 Fnr/非 ArcAB 有氧/厌氧调节机制的分子模型。这项研究具有非常重要的意义，因为它将揭示一条新的调控途径的参与者，该途径调节 An 以及可能的其他牙周病原体对厌氧龈下微环境的适应。表征未定义的、潜在的新转录调控途径非常重要，因为所涉及的蛋白质可能作为未来药物开发的靶点。