Skare J T, Shang E S, Foley D M, Blanco D R, Champion C I, Mirzabekov T, Sokolov Y, Kagan B L, Miller J N, Lovett M A
Department of Microbiology and Immunology, UCLA School of Medicine 90024, USA.
J Clin Invest. 1995 Nov;96(5):2380-92. doi: 10.1172/JCI118295.
We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.
我们基于为分离梅毒螺旋体的外膜囊泡（OMV）所开发的方法，从伯氏疏螺旋体B31菌株中分离并纯化了OMV。纯化后的OMV表现出不同的孔蛋白活性，电导分别为0.6和12.6纳西门子，并且未检测到β - NADH氧化酶活性，分别表明其外膜来源以及不存在内膜污染。通过与Triton X - 114进行相分配来鉴定疏水蛋白。这些疏水膜蛋白中的大多数未被酰化，表明它们是跨外膜的蛋白。对棕榈酸酯标记的脂蛋白的鉴定显示，有几种在OMV中富集，有几种在原生质圆柱体内膜组分中富集，还有其他一些仅与内膜相关。随着B31菌株的连续体外培养，OMV的蛋白质组成发生了显著变化。使用对强毒株B31具有特异性的抗血清，我们在螺旋体表面鉴定了OMV抗原，并鉴定出其在OMV中的存在可能与兔莱姆病模型中的毒力和保护性免疫相关的蛋白质。这些与强毒株相关的跨外膜蛋白可能为莱姆病的发病机制提供新的见解。
