Table_5_The proteome of bacterial membrane vesicles in Escherichia coli—a time course comparison study in two different media.XLSX

IntroductionBacteria 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.
MethodsTo gain a better insight into bMVs, we investigated the composition and functionality of E. coli (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.
ResultsParticle 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.
ConclusionOur study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli 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.

引言
细菌定植于人体内外各处，例如皮肤、肠道或口腔，可发挥无害、有益甚至致病的作用。众所周知，细菌可分泌膜囊泡（membrane vesicles, MVs），与携带细胞外囊泡（extracellular vesicles, EVs）的真核细胞类似。多项研究表明，细菌膜囊泡（bacterial membrane vesicles, bMVs）在微生物组-宿主互作中发挥关键作用。然而，此类细菌膜囊泡的组成及其在不同培养条件下的功能，目前仍未被充分阐明。

方法
为深入探究细菌膜囊泡，我们针对大肠杆菌（E. coli, DSM 105380）的细菌膜囊泡展开研究，分析其在两种培养基——Lysogeny broth (LB) 与 RPMI 1640——不同生长阶段（迟缓期、对数期与稳定期）的组成与功能。我们分别在3个时间点（8 h、54 h与168 h），从两种培养基中分离得到细菌膜囊泡，采用超速离心法进行纯化，并通过纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）、冷冻电子显微镜（cryogenic electron microscopy, Cryo-EM）、常规透射电子显微镜（transmission electron microscopy, TEM）以及基于液相色谱-串联质谱的蛋白质组学（LC–MS/MS）进行分析。此外，我们检测了经细菌膜囊泡处理后人单核细胞系THP-1中的促炎细胞因子IL-1β与IL-8的表达水平。

结果
细菌膜囊泡的颗粒数量随培养时长增加而升高。不同时间点与培养条件下的细菌膜囊泡形态，在冷冻电子显微镜与常规透射电子显微镜下均较为相似。通过蛋白质组学分析，我们在两种培养基、所有时间点分离得到的细菌膜囊泡中，共鉴定出140种蛋白质，包括常见的细菌膜囊泡标记物OmpA与GroEL。此外，我们还检测到了培养条件特异性的蛋白质。用全部6组细菌膜囊泡处理THP-1细胞后，均可显著诱导IL-1β与IL-8的表达上调。

结论
本研究表明，培养基的选择与培养时长，可显著影响大肠杆菌细菌膜囊泡的数量与蛋白质组成。本研究的透射电子显微镜与冷冻电子显微镜结果证实，我们成功分离得到了完整的大肠杆菌细菌膜囊泡。在全部6种测试的生长条件下，均可稳定检测到大肠杆菌的常见蛋白质，包括OmpA、GroEL以及核糖体蛋白。此外，本研究的功能实验结果表明，从6组样本中分离得到的细菌膜囊泡均保留了其生物学功能，可诱导相似水平的细胞因子表达。