The Treponema denticola major sheath protein is predominantly periplasmic and has only limited surface exposure
The Treponema denticola major sheath protein is predominantly periplasmic and has only limited surface exposure
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DOI：
10.1128/iai.67.8.4072-4083.1999
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发表时间：
1999-08-01
影响因子：
3.1
通讯作者：
Radolf, JD
中科院分区：
文献类型：
--
作者：
Caimano, MJ;Bourell, KW;Radolf, JD
最近发现梅毒螺旋体基因组编码12个齿密螺旋体主鞘蛋白(MSP)的同源基因，这促使我们重新研究齿密螺旋体T多肽的细胞位置和拓扑结构。最初进行的实验是为了确定MSP是否在齿纹夜蛾外膜上或膜内形成阵列。负染和超薄切片的透射电子显微镜(EM)未能鉴定出典型的表层，而冷冻断裂的EM显示齿纹夜蛾外膜含有异质跨膜蛋白，但没有排列。相反，在轻度超声的密螺旋体释放的囊泡中观察到一种晶格状结构；结合EM和生化分析表明，这种结构就是肽聚糖球囊。随后用免疫电子显微镜(IEM)对MSP在齿状毛滴虫中进行定位。对阴性染色的整体支架的检查发现，在超声生物中存在大量的MSP。超薄切片完整的梅毒螺旋体的IEM也表明，抗原优势与外膜无关。最后，对包埋在琼脂糖凝胶微滴中的密螺旋体进行了免疫荧光分析，发现只有很小一部分MSP暴露在表面。总体而言，我们的发现挑战了人们普遍认为的MSP在齿纹夜蛾外膜内形成阵列的观点，相反，我们证明了它主要是周质，只有有限的表面暴露。这些发现也对我们进化理解MSP/TPR同源基因在螺旋体生理学和疾病发病机制中的作用(S)具有重要意义。
The recent discovery that the Treponema pallidum genome encodes 12 orthologs of the Treponema denticola major sheath protein (Msp) prompted us to reexamine the cellular location and topology of the T, denticola polypeptide. Experiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron microscopy (EM) of negatively stained and ultrathin-sectioned organisms failed to identify a typical surface layer, whereas freeze-fracture EM revealed that the T. denticola outer membrane contains heterogeneous transmembrane proteins but no array. In contrast, a lattice-like structure was observed in vesicles released from mildly sonicated treponemes; combined EM and biochemical analyses demonstrated that this structure was the peptidoglycan sacculus. Immunoelectron microscopy (IEM) subsequently was performed to localize Msp in T. denticola. Examination of negatively stained whole mounts identified substantial amounts of Msp in sonicated organisms. IEM of ultrathin-sectioned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analysis of treponemes embedded in agarose gel microdroplets revealed that only minor portions of Msp are surface exposed. Taken as a whole, our findings challenge the widely held belief that Msp forms an array within the T. denticola outer membrane and demonstrate, instead, that it is predominantly periplasmic with only limited surface exposure. These findings also have implications for our evolving understanding of the contribution(s) of Msp/Tpr orthologs to treponemal physiology and disease pathogenesis.