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

• Published in: PloS one Vol. 9; no. 5; p. e95137
• Main Authors: Nakao, Ryoma, Kikushima, Kenji, Higuchi, Hideo, Obana, Nozomu, Nomura, Nobuhiko, Bai, Dongying, Ohnishi, Makoto, Senpuku, Hidenobu
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15-05-2014; Public Library of Science (PLoS)
• Subjects: Antigens; Appendages; Bacteria; Bacterial Proteins - metabolism; Bacteriology; Beads; Binding; Binding, Competitive; Biology and Life Sciences; Buffers (chemistry); Cell culture; Cell Culture Techniques - methods; Cell Line, Tumor; Cell Membrane - metabolism; Chemical bonds; Denaturation; Detachment; E coli; Electron microscopy; Engineering and Technology; Environmental science; Epoxy resins; Escherichia coli; Fimbriae, Bacterial - metabolism; Gum disease; Health care; Hemoglobin; Humans; Infectious diseases; Magnetics; Medicine and Health Sciences; Membrane vesicles; Microbiological Techniques - methods; Microscopy; Microscopy, Electron; Neisseria gonorrhoeae; Neisseria meningitidis; Pathogens; Periodontal disease; Periodontitis; Periodontitis - microbiology; Pili; Porphyromonas gingivalis; Porphyromonas gingivalis - metabolism; Protein denaturation; Pseudomonas aeruginosa; Purification; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; Structural integrity; Surface chemistry; Ultracentrifugation; Vesicles; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0095137

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.