Physical Properties and Shifting of the Extracellular Membrane Vesicles Attached to Living Bacterial Cell Surfaces.

Bacterial cells release nanometer-sized extracellular membrane vesicles (MVs) to deliver cargo molecules for use in mediating various biological processes. However, the detailed processes of transporting these cargos from MVs to recipient cells remain unclear because of the lack of imaging techniques to image nanometer-sized fragile vesicles in a living bacterial cell surface. Herein, we quantitatively demonstrated that the direct binding of MV to the cell surface significantly promotes hydrophobic quorum-sensing signal (C16-HSL) transportation to the recipient cells. Moreover, we analyzed the MV-binding process in the Paracoccus denitrificans cell surface using high-speed atomic force microscopy phase imaging. Although MV shapes were unaltered after binding to the cell surface, the physical properties of a group of single MV particles were shifted. Additionally, the phase shift values of MVs were higher than that of the cell's surfaces upon binding, whereas the phase shift values of the group of MVs were decreased during observation. The shifting physical properties occurred irreversibly only once for each MV during the observations. The decreasing phase shift values indicated alterations of chemical components in the MVs as well, thereby suggesting the dynamic process in which single MV particles deliver their hydrophobic cargo into the recipient cell. Compared to the increasing knowledge about MV release mechanisms from donor cells, the mechanism by which recipient cells receive cargo from MVs remains unknown. Herein, we have successfully imaged single MV-binding processes in living bacterial cell surfaces. Accordingly, we confirmed the shift in the MV hydrophobic properties after landing on the cell surface. Our results showed the detailed states and the attaching process of a single MV into the cell surface and can aid the development of a new model for MV reception into Gram-negative bacterial cell surfaces. The insight provided by this study is significant for understanding MV-mediated cell-cell communication mechanisms. Moreover, the AFM technique presented for nanometer-scaled mapping of dynamic physical properties alteration on a living cell could be applied for the analyses of various biological phenomena occurring on the cell surface, and it gives us a new view into the understanding of the phenotypes of the bacterial cell surface.