凝胶微滴中的梅毒螺旋体：一种用于BamA（TP0326）拓扑分析和稀有外膜蛋白定位的方法。
Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP0326) and Localization of Rare Outer Membrane Proteins.
作者信息
Luthra Amit, Anand Arvind, Radolf Justin D
机构信息
Department of Medicine, University of Connecticut Health, Farmington, CT, 06030, USA.
Department of Pediatrics, University of Connecticut Health, Farmington, CT, 06030, USA.
出版信息
Methods Mol Biol. 2015;1329:67-75. doi: 10.1007/978-1-4939-2871-2_6.
The noncultivable spirochete Treponema pallidum subspecies pallidum (T. pallidum) is the etiological agent of venereal syphilis. In contrast to the outer membranes (OMs) of gram-negative bacteria, the OM of T. pallidum lacks lipopolysaccharide, contains a paucity of integral membrane proteins, and is extremely labile. The lability of the T. pallidum OM greatly hinders efforts to localize the bacterium's rare outer membrane proteins (OMPs). To circumvent this problem, we developed the gel microdroplet method in which treponemes are encapsulated in porous agarose beads and then probed with specific antibodies in the absence or presence of low concentrations of the non-ionic detergent Triton X-100. To demonstrate the general utility of this method for surface localization of any T. pallidum antigen, herein we describe a protocol for immunolabeling of encapsulated treponemes using antibodies directed against the β-barrel and POTRA domains of TP0326, the spirochete's BamA ortholog.
不可培养的梅毒螺旋体苍白亚种（梅毒螺旋体）是性病梅毒的病原体。与革兰氏阴性菌的外膜（OM）不同，梅毒螺旋体的OM缺乏脂多糖，整合膜蛋白稀少，且极其不稳定。梅毒螺旋体OM的不稳定性极大地阻碍了定位该细菌罕见外膜蛋白（OMP）的努力。为规避这一问题，我们开发了凝胶微滴法，即将梅毒螺旋体包裹在多孔琼脂糖珠中，然后在不存在或存在低浓度非离子去污剂Triton X-100的情况下用特异性抗体进行检测。为证明该方法对任何梅毒螺旋体抗原表面定位的通用性，在此我们描述一种使用针对TP0326（该螺旋体的BamA直系同源物）的β桶和POTRA结构域的抗体对包裹的梅毒螺旋体进行免疫标记的方案。
Zotero 插件