Katona L I, Beck G, Habicht G S
Department of Pathology, State University of New York, Stony Brook 11794-8691.
Infect Immun. 1992 Dec;60(12):4995-5003. doi: 10.1128/iai.60.12.4995-5003.1992.
Borrelia burgdorferi resembles gram-negative bacteria in having both cellular and outer membranes. We previously showed that a lipopolysaccharide (LPS)-like material could be extracted from B. burgdorferi with phenol-chloroform-petroleum ether (PCP). The PCP extract of B. burgdorferi exhibited biological activity in several in vitro assays (e.g., mitogenicity, pyrogenicity, and cytokine release). These activities suggested the presence of endotoxin. The PCP extract of B. burgdorferi, however, also contained a small amount of protein. Preliminary studies showed that monoclonal antibody prepared against this protein inhibited the mitogenic activity of the PCP extract toward murine spleen cells. The current study was therefore undertaken to characterize this protein and to establish methods for its separation from the LPS. The PCP-extracted protein consisted of a single, low-molecular-weight lipoprotein (apparent M(r), 10,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (SDS-PAGE). By protein analysis, it accounted for 2% of the dry weight of defatted cells, thus making it a major constituent of the spirochete. It was purified from the LPS by initial extraction into 10% Triton X-100 followed by immunoaffinity chromatography in the presence of detergent. On removal of the LPS, the purified lipoprotein formed aggregates stable to SDS-PAGE which were detectable on Western blots (immunoblots) probed with either the monoclonal antibody or polyclonal antiserum. From a plot of the aggregate molecular weight versus aggregate size, a monomer molecular weight of 7,500 was obtained. Indirect immunofluorescence with the monoclonal antibody showed that the lipoprotein was exposed at the surface of the spirochete in only a small percentage of cells. The lipoprotein was present in several strains of B. burgdorferi but absent in other Borrelia spp., treponemes, and gram-negative human pathogens, indicating species specificity.
伯氏疏螺旋体在拥有细胞膜和外膜方面类似于革兰氏阴性菌。我们之前表明，可用苯酚 - 氯仿 - 石油醚（PCP）从伯氏疏螺旋体中提取出一种类似脂多糖（LPS）的物质。伯氏疏螺旋体的PCP提取物在多种体外试验中表现出生物活性（如促有丝分裂活性、致热活性和细胞因子释放）。这些活性提示存在内毒素。然而，伯氏疏螺旋体的PCP提取物也含有少量蛋白质。初步研究表明，针对该蛋白质制备的单克隆抗体抑制了PCP提取物对小鼠脾细胞的促有丝分裂活性。因此开展了当前这项研究来鉴定该蛋白质，并建立将其与LPS分离的方法。PCP提取的蛋白质由一种单一的低分子量脂蛋白组成（通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）测定的表观分子量为10,000）。通过蛋白质分析，它占脱脂细胞干重的2%，因此是螺旋体的主要成分。首先将其提取到10% Triton X - 100中，然后在有去污剂存在的情况下进行免疫亲和层析，从而从LPS中纯化出来。去除LPS后，纯化的脂蛋白形成了对SDS - PAGE稳定的聚集体，在用单克隆抗体或多克隆抗血清进行探测的蛋白质印迹（免疫印迹）上可检测到。从聚集体分子量与聚集体大小的关系图中，得出单体分子量为7,500。用单克隆抗体进行的间接免疫荧光显示，仅在一小部分细胞中该脂蛋白暴露于螺旋体表面。该脂蛋白存在于几种伯氏疏螺旋体菌株中，但在其他疏螺旋体属、密螺旋体以及革兰氏阴性人类病原体中不存在，表明具有种属特异性。
