Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagengrid.5254.6, Frederiksberg C, Denmark.
Section of Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo, Norway.
Microbiol Spectr. 2022 Oct 26;10(5):e0181922. doi: 10.1128/spectrum.01819-22. Epub 2022 Aug 30.
Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the and genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.
外膜囊泡（OMVs）是革兰氏阴性菌（包括引起猪传染性胸膜肺炎的胸膜肺炎放线杆菌）自发释放的，这种细菌会导致猪传染性胸膜肺炎，给全球养猪业造成巨大的经济损失。先前已经证明胸膜肺炎放线杆菌 OMVs 中含有 Apx 毒素和蛋白酶以及抗原蛋白。然而，其内容物的全面特性及其与宿主免疫细胞的相互作用尚未得到充分研究。了解胸膜肺炎放线杆菌 OMVs 的蛋白质组成和免疫调节能力有助于阐明其生物学功能，并促进基于 OMV 的应用的发展。在本研究中，我们全面表征了天然胸膜肺炎放线杆菌 OMVs 的蛋白质组。此外，我们对野生型菌株和三个突变株的 OMV 蛋白质组进行了定性和定量比较，其中相关基因被破坏以增加 OMV 的产生和/或产生缺乏超抗原 PalA 的 OMVs。此外，还对胸膜肺炎放线杆菌 OMVs 与猪肺泡巨噬细胞的相互作用进行了研究。我们的结果表明，天然 OMVs 由胸膜肺炎放线杆菌 MIDG2331 自发释放，似乎通过灭活或活亲本细胞刺激的猪肺泡巨噬细胞抑制了先天免疫反应。研究结果表明，OMVs 可能在胸膜肺炎放线杆菌感染的初始阶段在操纵猪的防御中发挥作用。由于其内在的佐剂和抗原性，细菌外膜囊泡（OMVs）作为人类和动物使用的潜在疫苗越来越受到关注。已经研究了从猪的重要呼吸道病原体胸膜肺炎放线杆菌中释放的 OMVs 用于疫苗开发。我们之前的研究表明，胸膜肺炎放线杆菌分泌含有多种免疫原性蛋白的 OMVs。然而，用这些囊泡免疫猪不能减轻胸膜肺炎放线杆菌攻毒引起的猪肺病变，这表明胸膜肺炎放线杆菌 OMVs 在宿主-病原体相互作用中发挥了难以捉摸的作用。在这里，我们表明胸膜肺炎放线杆菌分泌的 OMVs 的产量和蛋白质含量可以通过 删除 和 基因来改变。此外，我们证明胸膜肺炎放线杆菌 OMVs 可抑制胸膜肺炎放线杆菌细胞刺激的猪肺泡巨噬细胞的免疫反应，这表明胸膜肺炎放线杆菌可能用来逃避宿主防御的一种新机制。
