Anaerobic Regulatory Pathways in a Periodontopathogen

描述：牙周炎是龈下间隙组织的一种炎症性疾病，与龈下菌群向革兰氏阴性菌的急剧转变有关。我们重点研究了其中一种牙周病原体放线菌（放线菌）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）中定植。因此，Aa已经进化出了毒力机制，允许它在这两种环境之间转换。利用二维蛋白凝胶，我们已经证明厌氧生长诱导了数十种Aa蛋白的合成，包括白质毒素（一种假定的杀死中性粒细胞的毒力蛋白）。许多其他蛋白质在无氧条件下被抑制，但在有氧的情况下被诱导。通过在An中产生确定的突变，我们已经表明，大多数这些蛋白质的好氧/厌氧调节是由Fnr或ArcAB途径控制的，正如预期的那样。然而，有趣的是，至少有八种其他蛋白质的调节，包括白蛋白，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物，我们假设Aa基因的一个子集将由一个（或多个）转录调节途径控制，这些途径以前在Aa或任何其他细菌系统中都没有被描述过。在有氧和厌氧生长中不同合成的Aa基因的完整补体将使用DNA微阵列进行鉴定（Specific Aim I）。重要的是，我们还将确定一组氧反应基因，如白蛋白，由先前未确定的非fnr /非arca途径调节。在Specific Aim II中，将采用系统和全面的分子遗传学方法来鉴定这种潜在的新型调控途径和涉及的转录因子。最后，我们新发现的氧调节蛋白与氧化还原调控的Aa启动子的相互作用将被表征（Specific Aim III）。这将使我们能够开始开发牙周病原体中非fnr /非arcab好氧/厌氧调节机制的第一个分子模型。这项研究具有重要意义，因为它将揭示一种新的调节途径，调节An，以及其他牙周病原体对牙龈下厌氧微环境的适应。表征未定义的、潜在的新转录调控途径是重要的，因为涉及的蛋白质可能作为未来药物开发的靶标。

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.