Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains

• Published in: MicrobiologyOpen (Weinheim) Vol. 12; no. 5; pp. e1388 - n/a
• Main Authors: Haugsten, Helene R., Kristoffersen, Anne K., Haug, Trude M., Søland, Tine M., Øvstebø, Reidun, Aass, Hans C. D., Enersen, Morten, Galtung, Hilde K.
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01-10-2023; John Wiley and Sons Inc; Wiley
• Subjects: Adhesins, Bacterial; Alzheimer's disease; Bacteria; bacterial vesicle; Bacterial vesicles; Biosynthesis; Confocal microscopy; Cysteine Endopeptidases; Dentistry; Diameters; Extracellular Vesicles; Fibroblasts; Fibroblasts - microbiology; Flow cytometry; Fluorescent dyes; Fluorescent indicators; Genetic diversity; Genotype & phenotype; Gingipain; Gingipain Cysteine Endopeptidases; Gram-positive bacteria; Gum disease; Humans; Inflammation; Internalization; Lipids; Localization; Microscopy; Original; Pathogenesis; Periodontitis; Periodontitis - microbiology; Porphyromonas gingivalis; Proteins; Size distribution; Size exclusion chromatography; Strains (organisms); Substrates; Teeth; Transmission electron microscopy; Ultrafiltration; Vesicles; Virulence; Virulence factors; Porphyromonas gingivalis; bacterial vesicle; virulence factors
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.1388

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1‐28, and W83, were isolated through ultrafiltration and size‐exclusion chromatography. Vesicle size distribution was measured by nano‐tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg‐gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle‐like structures. bEV‐gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.
Bacterial extracellular vesicles (bEVs) from Porphyromonas gingivalis were isolated and characterized before uptake into oral fibroblast was visualized. bEVs from P. gingivalis displayed active gingipains that may have a role in the pathogenesis of periodontitis.