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

IF 3.9 3区生物学 Q2 MICROBIOLOGY

MicrobiologyOpen Pub Date : 2023-10-16 DOI:10.1002/mbo3.1388

Helene R. Haugsten, Anne K. Kristoffersen, Trude M. Haug, Tine M. Søland, Reidun Øvstebø, Hans C. D. Aass, Morten Enersen, Hilde K. Galtung

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Abstract

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.

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牙龈卟啉单胞菌细胞外小泡的分离、表征和成纤维细胞摄取

牙周炎是一种由细菌引起的炎症，是世界范围内一个严重的健康问题，因为炎症会损害牙齿的支撑组织，并可能导致全身性疾病。牙龈卟啉单胞菌被认为是一种重要的牙周病原体，它会释放含有毒力因子（如牙龈蛋白酶）的细菌细胞外小泡（bEVs），这可能有助于牙周炎的发病机制。本研究旨在从三株牙龈卟啉单胞菌中分离和鉴定bEV，研究bEV对人类口腔成纤维细胞的摄取，并测定bEV的银杏蛋白酶活性。通过超滤和大小排阻色谱法从ATCC 33277、A7A1-28和W83三个菌株中分离出bEV。通过纳米跟踪分析（NTA）测量囊泡大小分布。透射电子显微镜用于bEV的可视化。流式细胞术用于检测bEVs，并使用对arg银杏蛋白酶特异性的底物通过酶测定法测量银杏蛋白酶活性。使用共聚焦显微镜观察bEVs进入口腔成纤维细胞的摄取。NTA显示bEV浓度为108至1011个粒子/mL，bEV直径为42至356 nm。TEM照片显示囊泡状结构。采用流式细胞术和酶法检测bEV银杏叶蛋白。用荧光染料标记的bEV孵育的成纤维细胞显示出与bEV内化一致的细胞内定位。总之，从牙龈卟啉单胞菌中成功分离和鉴定了bEVs，并记录了它们对人类口腔成纤维细胞的吸收。bEVs显示出活跃的牙龈蛋白酶，证明了它们起源于牙龈卟啉单胞菌，以及bEVs在牙周炎中的潜在作用。

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来源期刊

MicrobiologyOpen MICROBIOLOGY-

CiteScore

8.00

自引率

0.00%

发文量

审稿时长

20 weeks

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