Table_2_Bacterial growth stage determines the yields, protein composition, and periodontal pathogenicity of Porphyromonas gingivalis outer membrane vesicles.xls

IntroductionP. gingivalis (W83), as the keystone pathogen in chronic periodontitis, has been found to be tightly bound to systemic diseases. Outer membrane vesicles (OMVs) produced by P. gingivalis (W83) are thought to serve key functions in bacterial virulence and pathogenicity. This study aims to comprehend the biological functions of P. gingivalis OMVs isolated from different growth stages by comparing their physicochemical properties and pathogenicity.MethodsProtein composition was analyzed via isotope-labeled relative and absolute quantification (iTRAQ). Macrophage polarization and the expression of IL-6 and IL-1β were detected. The proliferation, migration, osteogenic differentiation, and IL-1b/NLRP3 expression of periodontal ligament stem cells (PDLSCs) were evaluated. P. gingivalis/P. gingivalis OMVs-induced periodontal models were also constructed in Sprague Dawley rats.ResultsThe protein composition of P. gingivalis OMVs isolated from different growth stages demonstrated obvious differences ranging from 25 KDa to 75 KDa. In the results of flow cytometry, we found that in vitro experiments the M1 subtype of macrophages was more abundant in the late-log OMVs and stationary OMVs groups which boosted the production of inflammatory cytokines more than pre-log OMVs. Compared to pre-log OMVs, late-log OMVs and stationary OMVs had more pronounced inhibitory effects on proliferation, migration, and early osteogenesis of PDLSCs. The NLRP3 inflammasome was activated to a larger extent in the stationary OMVs group. Micro-computed tomography (Micro CT), hematoxylin-eosin staining (HE), and tartrate acid phosphatase (TRAP) results showed that the periodontal damage in the stationary OMVs group was worse than that in the pre-log OMVs and late-log OMVs group, but almost equal to that in the positive control group (P. gingivalis).DiscussionIn general, both in vivo and in vitro experiments showed that late-log OMVs and stationary OMVs have more significant pathogenicity in periodontal disease.

牙龈卟啉菌（W83）作为慢性牙周炎的关键致病菌，已被证实与全身性疾病密切相关。牙龈卟啉菌（W83）产生的外膜囊泡（OMVs）被认为在细菌的致病性和侵袭性中发挥关键作用。本研究旨在通过比较不同生长阶段分离的牙龈卟啉菌OMVs的物理化学性质和致病性，来理解牙龈卟啉菌OMVs的生物学功能。方法：通过同位素标记的相对和绝对定量（iTRAQ）分析蛋白质组成。检测巨噬细胞极化和IL-6及IL-1β的表达。评估牙周韧带干细胞（PDLSCs）的增殖、迁移、成骨分化和IL-1b/NLRP3的表达。在Sprague Dawley大鼠中构建了牙龈卟啉菌/牙龈卟啉菌OMVs诱导的牙周模型。结果：从不同生长阶段分离的牙龈卟啉菌OMVs的蛋白质组成显示出从25 KDa到75 KDa的明显差异。在流式细胞术的结果中，我们发现体外实验中，M1亚型的巨噬细胞在晚对数OMVs和稳定OMVs组中更为丰富，这比预对数OMVs组增加了炎症细胞因子的产生。与预对数OMVs相比，晚对数OMVs和稳定OMVs对PDLSCs的增殖、迁移和早期成骨的抑制作用更为显著。在稳定OMVs组中，NLRP3炎症小体被激活的程度更大。微计算机断层扫描（Micro CT）、苏木精-伊红染色（HE）和酒石酸磷酸酶（TRAP）的结果显示，稳定OMVs组的牙周损伤程度优于预对数OMVs组和晚对数OMVs组，但几乎与阳性对照组（牙龈卟啉菌）相等。讨论：总的来说，无论是在体还是体外实验，晚对数OMVs和稳定OMVs在牙周病中的致病性更为显著。