Yu Mia S C, Chiang Dapi Menglin, Reithmair Marlene, Meidert Agnes, Brandes Florian, Schelling Gustav, Ludwig Christina, Meng Chen, Kirchner Benedikt, Zenner Christian, Muller Laurent, Pfaffl Michael W
Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich (TUM), Freising, Germany.
Institute of Human Genetics, University Hospital, LMU Munich, Munich, Germany.
Front Microbiol. 2024 Mar 6;15:1361270. doi: 10.3389/fmicb.2024.1361270. eCollection 2024.
Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown.
To gain a better insight into bMVs, we investigated the composition and functionality of (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.
Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.
Our study showed that the choice of medium and the duration of culturing significantly influence both bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact bMVs. Common proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.
细菌存在于人体的内外，如皮肤、肠道或口腔，它们在这些部位发挥着无害、有益甚至致病的作用。众所周知，细菌能够像真核细胞分泌细胞外囊泡（EVs）一样分泌膜囊泡（MVs）。多项研究表明，细菌膜囊泡（bMVs）在微生物群与宿主的相互作用中起着关键作用。然而，此类bMVs的组成及其在不同培养条件下的功能仍 largely未知。
为了更深入地了解bMVs，我们研究了来自溶原肉汤（LB）和RPMI 1640培养基的（DSM 105380）bMVs在整个生长阶段（延迟期、对数期和稳定期）的组成和功能。通过超速离心分离来自三个时间点（8小时、54小时和168小时）以及两种培养基（LB和RPMI 1640）的bMVs，并使用纳米颗粒跟踪分析（NTA）、低温电子显微镜（Cryo-EM）、传统透射电子显微镜（TEM）和基于质谱的蛋白质组学（LC-MS/MS）进行分析。此外，我们检测了bMV处理后人单核细胞系THP-1中促炎细胞因子IL-1β和IL-8的水平。
颗粒数量随接种时间增加。在每个时间点和条件下，Cryo-EM/TEM中的bMV形态相似。通过蛋白质组学，我们鉴定出140种蛋白质，如常见的bMV标志物OmpA和GroEL，它们存在于从两种培养基和所有时间点分离的bMVs中。此外，我们能够检测到生长条件特异性蛋白质。用所有六组的bMVs处理THP-1细胞导致IL-1β和IL-8表达显著升高。
我们的研究表明，培养基的选择和培养时间显著影响bMVs的数量和蛋白质组成。我们的TEM/Cryo-EM结果证明了完整bMVs的存在。在所有六个测试生长条件下都能一致鉴定出包括OmpA、GroEL和核糖体蛋白在内的常见蛋白质。此外，我们的功能分析表明，从六组中分离的bMVs保留了其功能，并导致相当的细胞因子诱导。
