H2S production and virulence of Treponema denticola

描述：这项应用是当前研究齿密螺旋体产生硫化氢和毒力之间关系的扩展。齿状毛滴虫已被确认为牙周疾病发生和发展的病原学微生物联盟中的重要成员。此外，破坏部位产生的挥发性硫化物的存在是牙周病的一个特征，其中硫化氢是这一家族的主要化合物。然而，用于产生硫化氢的代谢途径还没有被很好地理解。以往的研究表明，宿主细胞中存在的谷胱甘肽可能是产生硫化氢的底物。最近，我们已经证明齿纹夜蛾具有利用谷胱甘肽作为底物产生高水平H_2S的能力。我们已经确定了谷胱甘肽成功代谢所需的三种酶：γ-谷氨酰基转移酶(GGT)、半胱氨基甘氨酸酶(CGase)和半胱氨酸酶。GGT将谷胱甘肽转化为半胱氨酸和谷氨酸；CG酶催化半胱氨酸生成半胱氨酸和甘氨酸；半胱氨酸酶将L半胱氨酸消化为硫化氢、氨和丙酮酸。我们还证明了在动物模型中加入半胱氨酸和L半胱氨酸可引起肝细胞生长因子和牙周膜细胞的凋亡，谷胱甘肽是齿状毛滴虫病变形成所必需的。在这些和其他研究的基础上，提出了利用生化、分子遗传学和细胞生物学研究的三个具体目标，以解决这三种代谢酶在齿状囊致病中发挥关键作用的假说。具体目标1：对谷胱甘肽转化为硫化氢过程中涉及的基因和蛋白质进行分子表征。具体目标2：通过基因失活从谷胱甘肽中产生H_2S的齿状毛霉的酶途径的遗传学特征。特异目的3：在体内外检测H_2S/NH_3产生途径对齿纹夜蛾毒力的影响。这项应用旨在提供关于齿状毛霉从谷胱甘肽产生H_2S的酶途径的基础和关键信息。这些结果将阐明该酶途径的每个成员在齿纹夜蛾毒力中所起的作用机制。

DESCRIPTION: This application is an extension of current research examining the association between H2S production and virulence of Treponema denticola. T. denticola has been identified as an important member of a consortium of microorganisms as etiologic in the initiation and progression of periodontal diseases. In addition, the existence of volatile sulfur compounds produced at destructive sites is a characteristic feature of periodontal diseases, with H2S as a major compound in this family. Nevertheless, the metabolic pathways used to produce H2S are not well understood. Previous studies suggest that glutathione present in host cells can be a substrate for H2S production. Recently, we have shown that T. denticola has the capacity to utilize glutathione as a substrate to produce high levels of H2S. We have identified three enzymes that are required for the successful metabolism of glutathione: gamma-glutamyltransferase (GGT), cysteinylglycinase (CGase), and cystalysin. GGT converts glutathione into Cys-Gly and glutamic acid; CGase catalyze Cys-Gly to Cys and glycine; and cystalysin digests L-cysteine into H2S, ammonia, and pyruvate. We have also demonstrated that the addition of cystalysin and L-cysteine resulted in apoptosis of HGF and PDL cells and that glutathione was essential for lesion formation by T. denticola in an animal model. Based on these and other studies, three Specific Aims are proposed using biochemical, molecular genetic, and cell biologic studies to address the hypothesis that these three metabolic enzymes play a key role in T denticola pathogenesis. Specific Aim 1: To molecularly characterize the genes and proteins involved in converting glutathione to H2S. Specific Aim 2: To genetically characterize, by gene inactivation, the enzyme pathway of T. denticola that produces H2S from glutathione. Specific Aim 3: To measure the effects of the H2S/NH3 producing pathway on T. denticola virulence in vitro and in vivo. This application is designed to provide both seminal and critical information about the enzyme pathway of T. denticola to produce H2S from glutathione. The outcomes will elucidate the mechanisms of action that each member of the enzyme pathway plays in the virulence capacity of T. denticola.