Abstract
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.
Highlights
Among the various Gram-negative anaerobes that reside within the subgingival pockets, Porphyromonas gingivalis is regarded as a keystone pathogen in the development of periodontal diseases, due to its ability to orchestrate inflammation while being a minor constituent in the community of periodontal pockets [1]
membrane vesicles (MVs), we unexpectedly found that MVs of P. gingivalis appeared to bind to the beads after a 14-hour incubation at 4uC
When superparamagnetic beads (SBs)-NH2 was used for this assay, both the MVs and the fimbriae bound to the SB-NH2, as few proteins were observed in the lanes of the unbound fractions (Unbound, 1st and 5th)
Summary
Among the various Gram-negative anaerobes that reside within the subgingival pockets, Porphyromonas gingivalis is regarded as a keystone pathogen in the development of periodontal diseases, due to its ability to orchestrate inflammation while being a minor constituent in the community of periodontal pockets [1]. Development of a novel remedy against periodontal diseases would have a significant impact on improving general public health. Both Gram-negative and Gram-positive bacteria produce and release spherical, microstructural bodies called membrane vesicles (MVs) that range in size from 10 to 300 nm in diameter [9]. P. gingivalis MVs have been regarded as offensive weapons leading to tissue deterioration in periodontal disease [14]. The crucial role of P. gingivalis MVs in mucosal immunogenicity was shown by an in vivo study [17], suggesting the potential of MVs as non-replicating mucosal immunogens for periodontal disease vaccines
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