Aa Ltx Induced Cell Death

Aa Ltx 诱导细胞死亡

• 批准号: 6891030
• 负责人: Edward Thomas Lally
• 金额: $ 35.66万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891030
• 关键词: Actinobacillus actinomycetemcomitans; BCL2 gene /protein; actin binding protein; apoptosis; bacterial cytopathogenic effect; bacterial toxins; calcium flux; calpain; cell component structure /function; confocal scanning microscopy; cyclodextrins; cytolysis; endoplasmic reticulum; immunoprecipitation; membrane permeability; membrane potentials; mitochondria; necrosis; nuclear membrane; periodontitis; phosphorylation; protease inhibitor

DESCRIPTION: Actinobacillus actinomycetemcomitans is a facultative anaerobe that has been implicated in a variety of periodontal diseases. Its presence is most closely associated with localized juvenile periodontitis (LJP) where it is believed to be the etiologic agent. The role this organism plays in other periodontal diseases, however, is not entirely clear. While A. actinomycetemcomitans produces a number of virulence factors, a leukotoxin (Ltx) appears to be most associated with enabling this organism to establish an ecological niche and thereby achieve its pathogenic potential. This concept is supported by an increasing number of clinical studies that demonstrate a reproducible ability to culture strains with the high leukotoxic phenotype from patients with active LJP. The mechanism by which A. actinomycetemcomitans Ltx kills target cells is ambiguous since two mechanisms of eukaryotic cell death have been identified: necrosis and apoptosis. High concentrations (> 104 M) of Ltx induce very rapid target cell death with little morphologic or cytometric evidence of apoptosis. Nevertheless, cells exposed to low concentrations of Ltx exhibit an alteration in mitochondrial transmembrane permeability that is followed by: 1) dissipation of the mitochondrial transmembrane potential, 2) release of apoptosis-inducing factors, 3) generation of reactive-oxygen radicals, and 4) depletion of ATP stores. The earliest change that we have observed following the addition of Ltx to susceptible cells is a rise in intracellular calcium [Ca2+]; which is both immediate (within 10 sec) and sustained. The Ltx-induced Ca 2+ influx and apoptosis exhibit an identical dose-dependence and point to a mechanistic connection linking of Ca 2+ influx and the onset of apoptosis. The ability of a mAb to neutralize the cytolytic activity of the Ltx and to inhibit the [Ca 2+]; changes are a strong indication that the reaction begins with Ltx. The proposed investigations are designed to increase our understanding of Ltx cytolysis by: 1) exploring early steps that result in Ltx-induced [Ca2+] c increases in target cells; 2) identifying the origin(s) of the increased [Ca2+] c seen in Ltx-treated cells; 3) defining effects Ltx-induced [Ca 2+] c increases have on cellular organelles and 4) characterizing the function of the endoplasmic reticulum in Ltx-treated cells.

描述：伴放线放线杆菌是一种兼性厌氧菌，与多种牙周疾病有关。它的存在与局部青少年牙周炎 (LJP) 最密切相关，据信它是局部青少年牙周炎的病因。然而，这种微生物在其他牙周疾病中所起的作用尚不完全清楚。虽然 A. actinomycetemcomitans 产生许多毒力因子，但白细胞毒素 (Ltx) 似乎与该生物体建立生态位并从而实现其致病潜力最相关。这一概念得到了越来越多的临床研究的支持，这些研究证明了从活动性 LJP 患者中培养具有高白细胞毒性表型的菌株的可重复性。 A. actinomycetemcomitans Ltx 杀死靶细胞的机制尚不明确，因为已确定真核细胞死亡的两种机制：坏死和凋亡。高浓度 (> 104 M) 的 Ltx 会诱导非常快速的靶细胞死亡，几乎没有细胞凋亡的形态学或细胞计数证据。然而，暴露于低浓度 Ltx 的细胞表现出线粒体跨膜通透性的改变，随后：1) 线粒体跨膜电位的耗散，2) 细胞凋亡诱导因子的释放，3) 活性氧自由基的产生，以及 4 ) ATP 储备耗尽。在向易感细胞中添加 Ltx 后，我们观察到的最早变化是细胞内钙 [Ca2+] 的升高；这既是即时的（10秒内）又是持续的。 Ltx诱导的Ca 2+ 流入和细胞凋亡表现出相同的剂量依赖性，并表明Ca 2+ 流入和细胞凋亡的发生之间存在机械联系。 mAb 中和 Ltx 的细胞溶解活性并抑制 [Ca 2+] 的能力；变化强烈表明反应是从 Ltx 开始的。拟议的研究旨在通过以下方式增加我们对 Ltx 细胞溶解的理解：1）探索导致靶细胞中 Ltx 诱导的 [Ca2+]c 增加的早期步骤； 2) 确定 Ltx 处理细胞中 [Ca2+]c 增加的来源； 3) 确定 Ltx 诱导的 [Ca 2+] c 增加对细胞器的影响，4) 表征 Ltx 处理细胞中内质网的功能。