Shang E S, Skare J T, Exner M M, Blanco D R, Kagan B L, Miller J N, Lovett M A
Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California 90095, USA.
Infect Immun. 1998 Mar;66(3):1082-91. doi: 10.1128/IAI.66.3.1082-1091.1998.
The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable beta-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [3H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.
通过冷冻断裂分析表明，赫氏疏螺旋体的外膜含有低密度的跨膜外膜蛋白，这些蛋白尚未被分离或鉴定。在本研究中，我们报告了从赫氏疏螺旋体HS-1中纯化外膜囊泡（OMV）以及随后对其组成外膜蛋白的鉴定。通过在低pH值、低渗柠檬酸盐缓冲液中剧烈涡旋全菌释放赫氏疏螺旋体的外膜，并通过等密度蔗糖梯度离心进行分离。分离出的OMV表现出0.2至7.2 nS的孔蛋白活性，与其外膜来源一致。通过缺乏可测量的β-NADH氧化酶活性以及考马斯亮蓝染色未观察到原生质圆柱体相关蛋白，表明纯化的OMV相对不含内膜污染。大约60个蛋白点（其中一些是假定的等电异构体）具有25种不同的分子量，被鉴定为OMV富集物的组成成分。这些蛋白中的大多数也被证明对感染赫氏疏螺旋体的小鼠血清具有抗原性。其中七种抗原蛋白用[3H]棕榈酸标记，包括表面暴露的甘油磷酸二酯磷酸二酯酶、可变主要蛋白7和33以及15、17、38、42和67 kDa的蛋白，表明它们是外膜的脂蛋白成分。此外，用抗加氏疏螺旋体表面暴露的p66/Oms66孔蛋白的抗血清对OMV进行免疫印迹分析，证明存在p66（Oms66）外膜同源物。用蛋白酶K处理完整的赫氏疏螺旋体导致Oms66/p66同源物部分蛋白水解，表明它是表面暴露的。对OMV蛋白的这种鉴定和表征应有助于进一步研究蜱传回归热的发病机制和免疫。
