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

描述（由申请人提供）：在患病的牙周袋中，谷胱甘肽水平低于健康部位，H2S含量较高。人们认为，但尚未证实，这些硫醇化合物浓度的变化对牙周炎中所见的组织病理很重要。虽然致病口袋中谷胱甘肽和H2S水平受到干扰的机制尚不清楚，但共识是细菌起了关键作用。因此，我们一直在研究牙周病原体从谷胱甘肽中产生H2S的能力，因为这种代谢途径可以改变龈缝中这两种分子的水平。我们已经专注于主要的牙周病原体牙密螺旋体，并表明它可以通过三步酶途径（GTSP）将谷胱甘肽分解为H2S。我们的目标是阐明这些硫醇分解代谢途径的作用，以及它们使用和产生的代谢物，在牙周病理中。为此，我们进行了几项初步实验，得出以下相关结果：(1)牙牙草加谷胱甘肽能在体外诱导人牙龈成纤维细胞和牙周韧带细胞凋亡。(2)牙牙菌加谷胱甘肽能增加牙周膜细胞促炎细胞因子的合成。(3)在小鼠脓肿模型中，谷胱甘肽可使齿状霉引起的病灶增大。(4)齿龈草促进大鼠牙槽骨吸收，降低龈沟液谷胱甘肽水平。(5)最重要的是，我们最近在GTSP的第一个基因（ggt）上构建了denticola t缺失突变体。这个突变体不能将谷胱甘肽转化为H2S。因此，我们有独特的优势来验证细菌代谢引起的谷胱甘肽水平的降低，特别是T. denticola GTSP，以及伴随的H2S产生的增加，将在牙周炎中看到的宿主组织损伤中发挥关键作用。在Aim 1中，我们将使用野生型齿牙鼠和我们的？GTSP，特别是其H2S的产生，可以促进牙周细胞的凋亡，并调节宿主细胞在体外产生的细胞因子的水平和模式。最重要的是，T. denticola突变体将在两个动物模型中使用（Aim 2），以证明细菌降低谷胱甘肽和增加H2S的能力是导致体内组织/骨病理的关键。大鼠牙槽骨丢失模型也将用于测试三种GTSP抑制剂的能力，我们已经在体外鉴定了它们在体内限制牙齿弓形虫发病的能力。拟议研究的结果将首次证明细菌影响龈下间隙谷胱甘肽和H2S水平的能力

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.