A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.
A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.
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DOI：
10.1371/journal.pone.0095137
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Senpuku H
中科院分区：
文献类型：
--
作者：
Nakao R;Kikushima K;Higuchi H;Obana N;Nomura N;Bai D;Ohnishi M;Senpuku H
牙龈卟啉单胞菌的膜泡（MV）被认为是细菌的进攻性武器，导致牙周病组织恶化。因此，分离高度纯化的MVs是必不可少的，以更好地了解MVs在牙周炎进展中的病理生理作用。MV通常通过基于细菌培养物上清液的超离心的常规方法分离。然而，所得MV通常被从活细菌剪切的共沉淀细菌附属物污染。在这里，我们报告了牙龈卟啉单胞菌MV的一个有趣的特性-它们结合环氧基包被的超顺磁珠（SB-环氧树脂）的能力。在纯化期间收集的级分的分析揭示了五种测试的牙龈卟啉单胞菌染色剂的所有MV与SB-环氧树脂结合。相比之下，粗MV制剂中的游离菌毛不与SB-环氧树脂结合。使用温和变性缓冲液，SB-环氧树脂结合的MV容易从SB-环氧树脂解离。这些结果表明，由环氧树脂赋予的珠的表面化学是负责的结合，这是由非共价键介导的。通过电子显微镜证实分离的MV的结构完整性和纯度。分离的MV也导致细胞在生理相关浓度下从培养皿脱离。SB-环氧树脂与来自五种细菌物种的MV的混合物之间的竞争性结合的测定表明，仅牙龈卟啉单胞菌MV可以从混合物中选择性地消除。我们认为，这种新的方法能够有效的纯化和选择性消除牙龈卟啉单胞菌MV。
Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs–their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs.
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Keystone-Pathogen假设。
DOI：
10.1038/nrmicro2873
发表时间：
2012-10
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
通讯作者：
--
影响因子：
37.8
作者：
Desvarieux, M;Demmer, RT;Papapanou, PN
通讯作者：
Papapanou, PN
影响因子：
3.1
作者：
Dorn, BR;Dunn, WA;Progulske-Fox, A
通讯作者：
Progulske-Fox, A
影响因子：
3.6
作者：
Aldick, Thomas;Bielaszewska, Martina;Karch, Helge
通讯作者：
Karch, Helge
影响因子：
15.3
作者：
MANDRELL, RE;GRIFFISS, JM;MACHER, BA
通讯作者：
MACHER, BA