BORRELIA BURGDORFERI PHOSPHOLIPASE ACTIVITY

伯氏疏螺旋体磷脂酶活性

• 批准号: 2074649
• 负责人: Robert Gregory Cluss
• 金额: $ 10.96万
• 依托单位: MIDDLEBURY COLLEGE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-15 至 2000-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2074649
• 关键词: Borrelia; Lyme disease; bacterial proteins; enzyme activity; enzyme substrate; gene expression; hemolysin; inflammation; lipolysis; molecular cloning; nucleic acid sequence; phospholipase A2; phospholipids; protein purification; secretory protein; virulence

DESCRIPTION (Adapted from the applicant's abstract): Borrelia burgdorferi, the Lyme disease spirochete, causes a multi-systemic disease, which in its chronic stage typically includes an inflammatory arthritis. This condition persists in individuals with long-term infections, despite the presence of relatively few organisms. It is likely that a number of spirochete factors initiate and maintain this inflammatory process and that the host immune response contributes to its severity. A new etiology for inflammation is suggested by our preliminary research which demonstrates a B. burgdorferi phospholipase A2 (PLA2) activity. This enzyme is almost certainly involved in lipid metabolism in the spirochete. In addition, we hypothesize that the PLA2 is involved in fatty acid uptake from the host. The acquisition of these metabolites may damage cells and tissues and contributes to inflammation. The specific aims of the proposed project are to: (1) to continue ongoing efforts to purify a B. burgdorferi phospholipase PLA2 activity associated with a soluble cell fraction, (2) to biochemically characterize the activity with particular attention to identifying lipid substrates subject to degradation, and consider how these degradation products might possibly contribute to inflammation, (3) to examine how the physiological state of the spirochete and various external factors affect the expression of the PLA2 and (4) to determine if a secreted 25-27 kDa protein from B. burgdorferi has phospholipase or hemolytic activity. Initial characterization of PLA2 preparations partially purified by heparin affinity chromatography indicate that the enzyme is heat stabile, hydrolyses at least 4 different phospholipids, does not require Ca2+ or a reducing environment for activity, and is inhibited by indomethacin. Enzyme activity was retained in the concentrate following ultrafiltration through a 10 Kda membrane, and SDS-PAGE followed by silver staining suggests a molecular mass for the B. burgdorferi enzyme similar to that of phospholipases from a wide range of gram positive and gram negative bacteria. In addition, a secreted protein of 25-27 kDa will be examined for phospholipase and hemolytic activity. Since PLA2 activity has heretofore not been described in B. burgdorferi, this study will help to provide insight into how the bacterium assimilates lipids from the host, penetrates cell membranes, and initiates an inflammatory response.

描述（改编自申请人的摘要）：疏螺旋体 伯氏疏螺旋体（莱姆病螺旋体）会引起多系统感染 疾病，其慢性阶段通常包括炎症 关节炎。这种情况在长期患病的个体中持续存在 尽管存在相对较少的生物体，但仍然存在感染。这是 可能有许多螺旋体因素引发并维持这种情况 炎症过程和宿主免疫反应有助于其 严重程度。我们提出了一种新的炎症病因 初步研究表明伯氏疏螺旋体磷脂酶 A2 (PLA2) 活性。这种酶几乎肯定与脂质有关 螺旋体中的新陈代谢。此外，我们假设 PLA2 参与宿主的脂肪酸摄取。收购 这些代谢物可能会损害细胞和组织并导致 炎。拟议项目的具体目标是： (1) 继续努力纯化伯氏疏螺旋体磷脂酶 PLA2 与可溶性细胞组分相关的活性，(2) 生化 表征活性，特别注意识别脂质 基材会发生降解，并考虑这些降解是如何发生的 产品可能会导致炎症，(3) 检查如何 螺旋体的生理状态和各种外界因素 影响 PLA2 的表达，并 (4) 确定是否分泌 来自伯氏疏螺旋体的 25-27 kDa 蛋白质具有磷脂酶或溶血性 活动。 PLA2 制剂的初步表征部分 经肝素亲和层析纯化表明该酶为 热稳定，水解至少 4 种不同的磷脂，不 需要 Ca2+ 或还原性环境才能活动，并受到抑制 通过吲哚美辛。浓缩物中保留了酶活性 通过 10 Kda 膜超滤后，进行 SDS-PAGE 随后银染表明 B 的分子量。 伯氏疏螺旋体酶与多种磷脂酶相似 革兰氏阳性菌和革兰氏阴性菌。此外，还有一种分泌的 将检查 25-27 kDa 的蛋白质的磷脂酶和溶血性 活动。由于迄今为止 B 中尚未描述 PLA2 活性。 伯氏杆菌，这项研究将有助于深入了解如何 细菌吸收宿主的脂质，穿透细胞膜， 并引发炎症反应。