Anaerobic Regulatory Pathways in a Periodontopathogen

牙周病原菌的厌氧调节途径

• 批准号: 6915772
• 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/6915772
• 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：牙周炎是一种位于龈下缝隙组织的炎症性疾病，与龈下微生物区系向革兰氏阴性菌转变有关。我们重点研究了这些牙周病原体之一，放线杆菌伴生放线杆菌(AA)的毒力特性。这种细菌与局部侵袭性牙周炎和几种成人牙周疾病密切相关。AA是一种兼性厌氧菌，能够在厌氧微环境(如患病的牙周袋)和有氧环境(如血流)中定居。因此，AA进化出了毒力机制，使其能够在这两种环境之间转换。使用二维蛋白质凝胶，我们已经证明了厌氧生长诱导数十种AA蛋白质的合成，包括白毒素(一种假定的杀死中性粒细胞的毒性蛋白质)。许多其他蛋白质在厌氧条件下被抑制，但在氧气存在时被诱导。通过在AN中产生明确的突变，我们已经证明，正如预期的那样，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。然而，有趣的是，包括白毒素在内的至少8种其他蛋白质的调节不受FNR或ArcAB的控制。由于AA不编码任何其他细菌氧调节蛋白的重要同源蛋白，我们假设AA基因的一个子集将由一个(或多个)转录调控途径控制，这些途径以前在AA或任何其他细菌系统中都没有描述过。在有氧和无氧生长中差异合成的全部AA基因将使用DNA微阵列(特定目标I)进行鉴定。重要的是，我们还将确定一组氧反应基因，如白毒素，这些基因受以前未识别的非FNR/非ArcA途径(S)调控。在特定的目标II中，将采用系统和全面的分子遗传学方法来鉴定这一潜在的新的调控途径和涉及的转录因子。最后，我们将表征我们新发现的氧调节蛋白与氧化还原调节的AA启动子的相互作用(特定目标III)。这将使我们能够开始开发第一个分子模型来研究牙周病原体中非FNR/非ArcAB有氧/厌氧调节的机制。这项研究具有非常重要的意义，因为它将揭示参与者在一种新的调节途径中调节AN，可能还有其他牙周病原体，以适应厌氧的牙龈下微环境。确定未定义的、潜在的新的转录调控途径是重要的，因为所涉及的蛋白质可能成为未来药物开发的靶点。