BORRELIA BURGDORFERI PHOSPHOLIPASE ACTIVITY

描述(改编自申请人的摘要)：疏螺旋体 伯氏杆菌，莱姆病螺旋体，引起多系统 疾病，在其慢性阶段通常包括炎症性 关节炎。这种情况在长期患有癌症的患者中持续存在 感染，尽管微生物相对较少。它是 很可能是一些螺旋体因素引发和维持了这种情况 炎症过程和宿主免疫反应对其 严肃性。我们提出了一种新的炎症病因学 一种伯氏杆菌磷脂酶的初步研究 A2(磷脂酶A2)活性。这种酶几乎肯定与脂肪有关。 螺旋体的新陈代谢。此外，我们假设PLA2 参与宿主对脂肪酸的摄取。收购 这些代谢物可能会损害细胞和组织，并有助于 发炎。拟议项目的具体目标是：(1) 继续努力纯化伯氏杆菌磷脂酶PLA2 与可溶性细胞组分有关的活性，(2)生化活性 表征活性，特别注意识别脂类 容易降解的底物，并考虑这些降解是如何 产品可能会导致炎症，(3)检查如何 螺旋体的生理状态与各种外界因素 影响PLA2的表达和(4)确定是否分泌 伯氏杆菌25-27 kDa蛋白具有磷脂酶或溶血酶活性 活动。部分PLA2制剂的初步表征 肝素亲和层析纯化表明，该酶是 耐热，至少能分解4种不同的磷脂，不 需要钙离子或还原环境才能活动，并被抑制 用消炎痛。浓缩物中的酶活性保持不变 通过10KDA膜超滤后，并进行SDS-PAGE 随后的银染表明B的分子质量。 伯氏杆菌酶与范围广泛的磷脂酶相似 革兰氏阳性和革兰氏阴性细菌。此外，一个秘密的 将对25-27 kDa的蛋白质进行磷脂酶和溶血检测 活动。因为磷脂酶A2的活性到目前为止还没有在B. Burgdorferi，这项研究将有助于了解 细菌从宿主吸收脂类，穿透细胞膜， 并引发炎症反应。

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.