The composition and functionality of bacterial membrane vesicles (bMVs) in Escherichia coli – a time course comparison study in different media

Abstract Background Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial, e.g. as probiotic bacteria, or even pathogenic role, e.g. during a sepsis. Recent studies have shown 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. Methods To get more insights 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 3 time points from 2 media (LB_8h, LB_54h, LB_168h, RPMI_8h, RPMI_54h and RPMI_168h) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), EV flow-cytometry, 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-1 and IL-8 in the human monocyte cell line THP-1 by treatment with bMVs. Results Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. EV flow-cytometry showed a similarity of the common bMV markers OmpA + GroEL - and OmpA - GroEL + in each group. We found 140 proteins to be consistently expressed in the 6 groups with LC-MS/MS proteomics while we could also observe unique proteins throughout these treatments. Treatment of THP-1 cells with bMVs of all 6 groups lead to significantly high IL-1 and IL-8 expressions. Conclusions Our study showed that the choice of medium and the duration of culturing significantly influences E.coli bMV protein composition. Moreover, our flow-cytometry results indicate that different bMV subpopulations may be shed. Irrespective of the medium used, we observed an accumulation of E. coli bMVs over time, possibly due to increase of bacterial cells. Our cell culture experiments/functional assays imply that bMVs isolated from the 6 groups by ultracentrifugation retain their function and lead to comparable cytokine induction.