Evaluation of OMV Production and GFP Secretion Through OMVs

Ten microliters of the isolated OMVs or a culture of E. coli cells was analyzed via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining. OMV production was quantified as previously described (Schwechheimer and Kuehn, 2013) with some modifications. The OMV concentration was measured by quantifying the band at approximately 37 kDa in the SDS-PAGE gel using the Image J software (National Institutes of Health, Bethesda, MD, United States). The OMV production of each mutant was shown as relative value to WT.

Outer membrane vesicles were also quantified with a lipophilic dye FM4-64 according to a previously described method with minor modifications (McBroom et al., 2006; Manning and Kuehn, 2011; Klimentová and Stulík, 2015). Isolated OMVs were incubated with 5 μg/ml FM4-64 (Molecular Probes/Thermo Fisher, Waltham, MA, United States) in PBS (pH 7.5) for 20 min. Then, OMVs were measured at the excitation and emission wavelengths of 558 and 734 nm, respectively, using an INFINITE 200 PRO spectrofluorophotometer (TECAN, Switzerland). The OMV sample without staining by a lipophilic dye (FM4-64) or the lipophilic dye alone was used as the negative controls.

The His-tagged GFP included in OMV samples was analyzed via western blotting by first transferring the protein from a gel to an Immobilon-P membrane sheet (Merck Millipore, Billerica, MA, United States) using the semidry transfer method. The blot was hybridized with an anti-histidine-tag primary monoclonal antibody (Medical & Biological Laboratories Co., Nagoya, Japan) and then a secondary antibody, the anti-mouse immunoglobulin G (whole molecule)–alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, United States). The hybridization signals were detected using a BCIP-NBT Solution Kit for Alkaline Phosphatase Stain (Nacalai Tesque, Kyoto, Japan). The level of the target protein was quantified by analyzing the bands on the western blot band using densitometry. GFP secretion was quantified on the basis of the intensity of a GFP standard sample (50 μg/L) with a His-tag (Sino Biological Inc., Beijing, China). The GFP produced in each E. coli cell was also analyzed via western blotting. In the case of cell analysis, a 10 μl sample at OD₆₆₀ = 0.3 was loaded into each well of gel to include the same amount of cells.

The GFP-derived fluorescence of the OMV samples was observed at an excitation wavelength of 488 nm using a confocal laser scanning microscope (CLSM; DM6000B, Leica, Wetzlar, Germany) with the TCS SP8 software.