PDF(Pubmed)
Abstract：
UNASSIGNED: 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.
UNASSIGNED: To 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.
UNASSIGNED: 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.
UNASSIGNED: Our 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.
UNASSIGNED: To 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.
UNASSIGNED: 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.
UNASSIGNED: Our 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.
摘要：
■细菌栖息在人体内外，如皮肤，肠道或口腔，它们在那里发挥无害的作用，有益甚至致病作用。众所周知，细菌可以像具有细胞外囊泡（EV）的真核细胞一样分泌膜囊泡（MV）。一些研究表明，细菌膜囊泡（bMVs）在微生物组-宿主相互作用中起关键作用。然而,这些bMV的组成及其在不同培养条件下的功能在很大程度上仍然未知。
■为了更好地了解bMV，我们调查了来自培养基的大肠杆菌（DSM105380）bMVs的组成和功能。溶源肉汤（LB）和RPMI1640在整个不同的生长阶段（lag-，对数和固定阶段)。来自三个时间点的bMV(8小时，54h,和168h）和两种介质（LB和RPMI1640）通过超速离心分离，并使用纳米颗粒跟踪分析（NTA）进行分析，低温电子显微镜（Cryo-EM），常规透射电子显微镜（TEM）和基于质谱的蛋白质组学（LC-MS/MS）。此外，我们检测了bMV处理后人单核细胞系THP-1中的促炎细胞因子IL-1β和IL-8。
■颗粒数随接种期增加。在每个时间点和条件下，Cryo-EM/TEM中的bMV形态相似。使用蛋白质组学，我们鉴定出140种蛋白质,例如常见的bMV标记OmpA和GroEL，存在于从两种培养基和所有时间点分离的bMV中。此外,我们能够检测生长条件特异性蛋白。用所有六组的bMV处理THP-1细胞导致显著高的IL-1β和IL-8表达。
■我们的研究表明，培养基的选择和培养的持续时间显着影响大肠杆菌bMV数量和蛋白质组成。我们的TEM/Cryo-EM结果证明存在完整的大肠杆菌bMV。常见的大肠杆菌蛋白，包括OmpA,GroEL,和核糖体蛋白，可以在所有六个测试的生长条件下一致地识别。此外，我们的功能测定表明,从6组分离的bMV保留了其功能,并导致相当的细胞因子诱导.
■为了更好地了解bMV，我们调查了来自培养基的大肠杆菌（DSM105380）bMVs的组成和功能。溶源肉汤（LB）和RPMI1640在整个不同的生长阶段（lag-，对数和固定阶段)。来自三个时间点的bMV(8小时，54h,和168h）和两种介质（LB和RPMI1640）通过超速离心分离，并使用纳米颗粒跟踪分析（NTA）进行分析，低温电子显微镜（Cryo-EM），常规透射电子显微镜（TEM）和基于质谱的蛋白质组学（LC-MS/MS）。此外，我们检测了bMV处理后人单核细胞系THP-1中的促炎细胞因子IL-1β和IL-8。
■颗粒数随接种期增加。在每个时间点和条件下，Cryo-EM/TEM中的bMV形态相似。使用蛋白质组学，我们鉴定出140种蛋白质,例如常见的bMV标记OmpA和GroEL，存在于从两种培养基和所有时间点分离的bMV中。此外,我们能够检测生长条件特异性蛋白。用所有六组的bMV处理THP-1细胞导致显著高的IL-1β和IL-8表达。
■我们的研究表明，培养基的选择和培养的持续时间显着影响大肠杆菌bMV数量和蛋白质组成。我们的TEM/Cryo-EM结果证明存在完整的大肠杆菌bMV。常见的大肠杆菌蛋白，包括OmpA,GroEL,和核糖体蛋白，可以在所有六个测试的生长条件下一致地识别。此外，我们的功能测定表明,从6组分离的bMV保留了其功能,并导致相当的细胞因子诱导.
