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.
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.
Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
Ryoma
R
Nakao
Department of Science, The University of Tokyo, Tokyo, Japan.
Kenji
K
Kikushima
Department of Science, The University of Tokyo, Tokyo, Japan.
Hideo
H
Higuchi
Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan.
Nozomu
N
Obana
Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan.
Nobuhiko
N
Nomura
Department of Gerodontology, Graduate school of Tokyo Medical and Dental University, Tokyo, Japan.
Dongying
D
Bai
Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
Makoto
M
Ohnishi
Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
Hidenobu
H
Senpuku
D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D006801:Humans; D008280:Magnetics; D008828:Microbiological Techniques / Q000379:methods*; D008853:Microscopy; D008854:Microscopy, Electron; D010518:Periodontitis / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000378:metabolism*; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins