Multiple Functions of Glutathione Catabolism and H2S in T. denticola Pathogenesis

描述（由申请人提供）：在患病牙周袋中，谷胱甘肽水平低于健康部位，H2S含量较高。人们一直认为，但没有证明，这些硫醇化合物的浓度变化是重要的牙周炎中看到的组织病理学。虽然谷胱甘肽和H2S水平在患病口袋中受到干扰的机制尚不清楚，但共识是细菌起着关键作用。因此，我们一直在研究牙周病原体从谷胱甘肽产生H2S的能力，因为这样的代谢途径可以改变这两种分子在龈沟中的水平。我们已经集中在主要的牙周病原体齿垢密螺旋体，并已表明以前，它可以分解代谢谷胱甘肽H2S通过三步酶途径（GTSP）。我们的目标是阐明这种巯基分解代谢途径的作用，以及它们使用和产生的代谢产物，在牙周病理学。为此，我们进行了几个初步的实验，得到了以下有关结果：（1）T.实验结果表明，外源性谷胱甘肽可诱导体外培养的人牙龈成纤维细胞和牙周膜细胞凋亡。(2)T.在体外，加谷胱甘肽。(3)Glucosidase可加重T.在小鼠脓肿模型中的齿垢。(4)T.齿垢促进大鼠牙槽骨吸收并降低龈沟液谷胱甘肽水平。(5)最重要的是，我们最近建造了一个T。在GTSP的第一个基因（ggt）中的齿垢缺失突变体。该突变体不能将谷胱甘肽转化为H2S。因此，我们处于独特的地位，以测试这一假设，即谷胱甘肽水平的下降，由细菌代谢，特别是T。齿垢GTSP，以及伴随的H2S产生的增加将在牙周炎中观察到的宿主组织损伤中起关键作用。在目标1中，我们将使用野生型T。齿垢和我们的？ggt突变体，以证明GTSP，特别是其产生的H2S，增强牙周细胞的凋亡，并在体外调节宿主细胞产生的细胞因子的水平和模式。最重要的是，T。将在两种动物模型中使用Denticola突变体（目的2），以证明细菌降低谷胱甘肽和增加H2S的能力对于体内引起组织/骨病理学是至关重要的。牙槽骨丢失的大鼠模型也将用于测试我们已经在体外表征的三种GTSP抑制剂限制T。体内龋齿发病机制。拟议中的研究结果将首次证明，细菌能够影响龈下裂隙中谷胱甘肽和H2S水平 是牙周组织损伤的重要因素。尽管实验仅用T.结果将建立一个新的范例，即牙周细菌的生理学，单独或协同作用，可以扰乱硫醇分子的稳态，导致疾病的病理。通过抑制细菌分解代谢途径重建正常牙周袋代谢物水平可能是减少牙周炎宿主损害的创新策略。

DESCRIPTION (provided by applicant): In diseased periodontal pockets, glutathione levels are lower than in healthy sites and the amount of H2S is higher. It has been assumed, but not proven, that changes in the concentrations of these thiol-compounds are important for the tissue pathology seen in periodontitis. Although the mechanism by which the levels of glutathione and H2S are perturbed in diseased pockets is unknown, the consensus is that bacteria play a key role. Thus, we have been studying the ability of periodontal pathogens to produce H2S from glutathione since such a metabolic pathway could alter the levels of these two molecules in the gingival crevice. We have focused on the major periodontal pathogen Treponema denticola and have shown previously that it can catabolize glutathione to H2S via a three step enzymatic pathway (GTSP). Our goal is to elucidate the role of such thiol catabolic pathways, and the metabolites they use and produce, in periodontal pathology. To this end, we have conducted several preliminary experiments with the following relevant results: (1) T. denticola plus glutathione can induce apoptosis in human gingival fibroblasts and periodontal ligament cells in vitro. (2) Periodontal ligament cell synthesis of proinflammatory cytokines is increased by T. denticola plus glutathione in vitro. (3) Glutathione exacerbates the lesion size caused by T. denticola in a mouse abscess model. (4) T. denticola enhances alveolar bone resorption in rats and decreases gingival crevicular fluid glutathione levels. (5) Most significantly, we recently constructed a T. denticola deletion mutant in the first gene (ggt) of the GTSP. This mutant cannot convert glutathione into H2S. Thus, we are uniquely positioned to test the hypothesis that the decrease in glutathione levels by bacterial metabolism, specifically the T. denticola GTSP, and the accompanying increase in H2S production will play key roles in the host tissue damage seen in periodontitis. In Aim 1, we will use wild type T. denticola and our ?ggt mutant to demonstrate that the GTSP, particularly its generation of H2S, enhances apoptosis in periodontal cells and modulates the levels and pattern of cytokines produced by host cells in vitro. Most importantly, the T. denticola mutant will be used in two animal models (Aim 2) to prove that a bacterium's ability to lower glutathione and increase H2S is critical for causing tissue/bone pathology in vivo. The rat model of alveolar bone loss will also be used to test the ability of three GTSP inhibitors, which we have already characterized in vitro, to limit T. denticola pathogenesis in vivo. The outcomes of the proposed studies will prove, for the first time, that a bacterium's ability to influence glutathione and H2S levels in the subgingival crevice is a significant contributor to periodontal tissue damage. Although the experiments are only being done with T. denticola, the results would establish a new paradigm whereby the physiology of periodontal bacteria, singly or synergistically, could perturb thiol-molecule homeostasis to cause disease pathology. Re-establishing normal periodontal pocket metabolite levels pharmacologically by inhibiting a bacterial catabolic pathway could be an innovative strategy to reduce host damage in periodontitis.