Table_1_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 细菌定植于人体体表及体内，例如皮肤、肠道或口腔，可发挥无害、有益乃至致病的作用。众所周知，细菌可如真核细胞分泌细胞外囊泡（extracellular vesicles, EVs）一般，分泌膜囊泡（membrane vesicles, MVs）。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，目前仍尚未完全明晰。
方法 为更深入地解析细菌膜囊泡，本研究针对大肠杆菌（E. coli, DSM 105380）的膜囊泡，探究其在不同生长阶段（迟缓期、对数生长期及稳定期）下，于LB培养基（Lysogeny broth, LB）与RPMI 1640培养基中产生的膜囊泡的组成与功能。研究分别在3个时间点（8 h、54 h及168 h），采用两种培养基（LB与RPMI 1640），通过超速离心分离细菌膜囊泡，并借助纳米颗粒追踪分析（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（LC–MS/MS）的蛋白质组学分析对其进行检测。此外，本研究还检测了经细菌膜囊泡处理后人单核细胞系THP-1（human monocyte cell line THP-1）中促炎细胞因子（pro-inflammatory cytokines）IL-1β与IL-8的表达水平。
结果 随培养时长延长，细菌膜囊泡的颗粒数量逐渐增加。在各时间点与培养条件下，细菌膜囊泡的冷冻电子显微镜/透射电子显微镜形态均保持一致。通过蛋白质组学分析，本研究在两种培养基来源、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志物OmpA与GroEL。此外，本研究还检测到了生长条件特异性蛋白。将6组不同来源的细菌膜囊泡分别处理THP-1细胞后，均可显著诱导IL-1β与IL-8的高表达。
结论 本研究表明，培养基的选择与培养时长，均会显著影响大肠杆菌膜囊泡的产量与蛋白质组成。透射电子显微镜/冷冻电子显微镜结果证实，本研究分离得到的大肠杆菌膜囊泡结构完整。包括OmpA、GroEL及核糖体蛋白在内的大肠杆菌常见蛋白质，在全部6种测试培养条件下均可被稳定鉴定。此外，功能实验结果显示，6组来源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。