Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum
Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum
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DOI：
10.1128/iai.00834-10
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发表时间：
2010-12-01
影响因子：
3.1
通讯作者：
Radolf, Justin D.
中科院分区：
文献类型：
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作者：
Cox, David L.;Luthra, Amit;Radolf, Justin D.
梅毒螺旋体与兔和人梅毒血清中存在的抗体反应很差，这是由于其外膜中缺乏蛋白质的特性。为了更好地理解梅毒螺旋体的“隐形致病性”的基础，我们使用了双标记，3步扩增试验，其中包封在凝胶微滴中的密螺旋体与梅毒血清平行与抗FlaA抗体进行探测。一小部分（约5 - 10%）但可重现的完整密螺旋体结合IgG和/或IgM抗体。三条证据支持表面抗原可能是β-桶形成外膜蛋白（OMP）的观点：（i）未观察到抗类脂（VDRL）抗体的表面标记，（ii）免疫印迹分析证实了先前的结果，表明T.苍白球糖脂不具有免疫反应性，和（iii）标记的完整的生物体没有明显的蛋白酶K（PK）治疗的影响。用这种方法，我们还表明，TprK（TP 0897），一个广泛研究的候选OMP，和TP 0136，脂蛋白最近报道的表面暴露，都是周质。与免疫标记研究一致，还发现TprK缺乏两亲性，这是β-桶形成蛋白的特征性质。使用一个结合了亚细胞定位和β-桶结构预测工具的共识计算框架，我们生成了候选稀有OMP的排名组，预测的T。苍白球外膜蛋白质组（OMPeome），我们假设包括我们的增强凝胶微滴检测表面暴露的分子。除了强调梅毒螺旋体的表面抗原性非常差，我们的研究结果有助于解释梅毒感染的特点病原体和宿主防御之间的复杂和不断变化的平衡。
Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochete's "stealth pathogenicity," we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely beta-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of beta-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and beta-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochete's remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.