BORRELIA BURGDORFERI PHOSPHOLIPASE ACTIVITY

描述（改编自申请人摘要）：疏螺旋体

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.