AA LTX INDUCED CELL DEATH

AA LTX 诱导细胞死亡

• 批准号: 2897158
• 负责人: Edward Thomas Lally
• 金额: $ 26.09万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2897158
• 关键词: Actinobacillus actinomycetemcomitans; antisense nucleic acid; apoptosis; bacterial cytopathogenic effect; bacterial toxins; biological signal transduction; cytolysins; cytotoxicity; flow cytometry; gene expression; gingiva; host organism interaction; human tissue; integrins; lymphocyte; natural killer cells; neutrophil; nucleic acid sequence; pulsed field gel electrophoresis; receptor expression; toxin metabolism

Actinobacillus actinomycetemcomitans(Aa), a major pathogen in certain human periodontal diseases, elaborates a potent leukotoxin (Ltx). This polypeptide kills human neutrophils, monocytes and T lymphocytes and may impair host antibacterial resistance by destroying or perturbing these cells in infected tissues. Results of current studies indicate that the toxin disrupts the permeability barrier of the plasma membrane of susceptible cells. This results in a 2+rapid rise in Ca (and probably other ions) levels within the cell which ultimately leads to cell death. This proposal will determine the molecular mechanism(s) for this observation. Two separate lines of experimentation form the basis for this proposal: l) Ltx-treated lymphocytes and NK cells exhibit many of the morphological characteristics of apoptosis; and 2) the Ltx receptor is a beta2 integrin, a molecule which when stimulated is capable of elevating cytosolic 2+Ca levels. In Specific Aim #1, Ltx-treated cells will be evaluated by DNA degradation, macromolecular synthesis, and FACS-associated morphologic alterations to determine if programmed cell death is occurring. Specific Aim 2 will focus on the expression of genes known to be involved in programmed cell death such as Bc1-2, Bc1-x, ICE, Bax, CPP32 and p53 to determine a molecular mechanism for cell death. Specific Aim 3 proposes experiments to define the nature of the toxin/receptor interaction. We envision that the first contact that the Ltx contact on a cell is a (32 integrin cell surface molecule. It is our goal to define these earliest consequences of this interaction such as determine the signal 2+pathway that is activated and the relationship to elevated cytosolic Ca +2 levels. Finally Specific Aim 4 will identify genes which are unique to Ltx- mediated apoptosis. It now emerging that the process of apoptosis involves different pathways when different agents are used to initiate the process. From these experiments we will have a better understanding of the pathway that is utilized by the toxin to kill cells. These studies provide fundamental insights into the pathobiology of the Aa Ltx and will bring us closer to understanding of its role in human periodontal infections on a molecular level. In addition, detailed studies on the action of the Aa Ltx will contribute to a better understanding of the biology of other membrane-active bacterial cytolynsins.

伴放线放线杆菌（Aa）是一种主要病原菌 某些人类牙周疾病，精心制作出一种强效白细胞毒素 （Ltx）。该多肽可杀死人类中性粒细胞、单核细胞和 T 淋巴细胞并可能通过以下方式损害宿主的抗菌耐药性 破坏或扰乱受感染组织中的这些细胞。 结果 目前的研究表明，该毒素会破坏 易感细胞质膜的渗透屏障。 这导致 Ca（可能还有其他离子）快速上升 2+ 细胞内的水平最终导致细胞死亡。这 提案将确定其分子机制 观察。两条独立的实验线构成了基础 对于该提案：l) Ltx 处理的淋巴细胞和 NK 细胞表现出 细胞凋亡的许多形态学特征；和 2） Ltx 受体是一种 β2 整合素，当受到刺激时，该分子会 能够提高细胞质 2+Ca 水平。在具体目标#1中， Ltx处理的细胞将通过DNA降解进行评估， 大分子合成和FACS相关形态学 改变以确定程序性细胞死亡是否正在发生。 具体目标 2 将重点关注已知基因的表达 参与程序性细胞死亡，例如 Bc1-2、Bc1-x、ICE、Bax、 CPP32 和 p53 确定细胞死亡的分子机制。 具体目标 3 提出通过实验来定义 毒素/受体相互作用。我们预计第一次接触 细胞上的 Ltx 接触点是 (32 整合素细胞表面分子。 我们的目标是定义这一事件的最早后果 相互作用，例如确定信号 2+ 路径，即 激活以及与胞质 Ca +2 水平升高的关系。 最后，具体目标 4 将识别 Ltx- 特有的基因 介导细胞凋亡。现在发现细胞凋亡过程 当使用不同的药物时涉及不同的途径 启动该过程。 从这些实验中我们将得到更好的结果 了解毒素杀死病毒的途径 细胞。这些研究提供了对以下问题的基本见解： Aa Ltx 的病理生物学将使我们更接近 了解其在人类牙周感染中的作用 分子水平。此外，还对行动的详细研究 Aa Ltx 将有助于更好地理解 其他膜活性细菌溶细胞素。