Cox D L, Akins D R, Bourell K W, Lahdenne P, Norgard M V, Radolf J D
Division of Sexually Transmitted Diseases Laboratory Research, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7973-8. doi: 10.1073/pnas.93.15.7973.
We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.
我们采用了新颖的免疫荧光策略来证明伯氏疏螺旋体的外表面蛋白（Osp）A、B和C在表面的暴露程度有限，这一发现与这些抗原仅存在于表面的主流观点相矛盾。当将抗OspA或抗OspB抗体添加到活动的菌体中时，观察到轻度标记，而当对同一张玻片进行甲醇固定并重新检测时，则观察到强烈荧光。当将包裹在琼脂糖珠（凝胶微滴）中的螺旋体与针对这两种相同抗原的抗体一起孵育时，也观察到适度标记。这与在存在0.06% Triton X-100（可选择性去除外膜）的情况下检测包裹的螺旋体时观察到的强烈荧光形成对比。用蛋白酶K（PK）处理包裹的螺旋体可消除表面标记。然而，在存在去污剂的情况下，用PK处理过的螺旋体与抗OspA或抗OspB抗体孵育后会发出强烈荧光，这证实了大量表面下Osp抗原的存在。在过夜孵育后重新检测PK处理过的螺旋体时，再次检测到适度的表面标记，这一结果与假定的将脂蛋白转运到螺旋体表面的分泌装置的存在相一致。最后，对表达OspC的伯氏疏螺旋体菌株297进行的实验表明，尽管该抗原是分离的外膜的主要成分，但在螺旋体表面几乎检测不到。我们提出了一个伯氏疏螺旋体分子结构模型，有助于解释慢性莱姆病期间螺旋体的持续存在。
