Figure 1.
MVs visualized by TEM in pathogenic bacteria.
HPF-FS sections correspond to the three pathogenic strains grown in solid media: (A) N. gonorrhoeae, (B) Pseudomonas PAO1 and (C) A. baumannii. O-IMVs (marked with arrows) are observed in the extracellular matter of the three pathogenic bacteria. O-IMVs in A and B clearly show a double bilayer, which exhibits the same staining profile as OM and PM from the respective cells. The O-IMV inner membrane encloses a material similar to that seen in the cytoplasm of the respective cells. OM: outer membrane; PM: plasma membrane; CC: cytoplasmic content. Bars 200 nm.
Figure 2.
An O-IMV being released from the surface of a N. gonorrhoeae cell.
(A) The TEM micrograph provides a view of an O-IMV at the moment of its formation, where the outer membrane (OM) is being extruded, dragging along the plasma membrane (PM) and a portion of the cytoplasmic content (CC). (B) The same image as A but with the cell envelope outlined to highlight the formation and structure of the O-IMV. Bars, 200 nm.
Figure 3.
TEM micrographs from HPF-FS sections of MVs isolated from (A) N. gonorrhoeae, (B) Pseudomonas PAO1 and (C) A. baumannii. O-IMVs observed in MV preparations from the three strains have certain features in common: all are surrounded by an external bilayer, probably corresponding to the outer membrane (OM) of the cell, and contain an inner membrane, probably corresponding to the plasma membrane (PM) of the cell, which entraps a high electron-dense material. In the image of O-IMVs from A. baumannii the putative peptidoglican layer (PG) can be seen. Bars 100 nm.
Figure 4.
Cryo-TEM visualization of O-IMVs in pathogenic bacteria.
Cryo-electron micrographs showing whole plunge-frozen cells from three pathogenic bacteria, and their derived O-IMVs: (A) N. gonorrhoeae, (B) Pseudomonas PAO1, and (C) A. baumannii. Whole cells with well-defined envelopes are observed in A and B (large black squares show a magnified area of cell envelopes). The new O-IMVs in the three analyzed samples exhibit the same double layer as cells, and are filled with an electron-dense material similar to that seen in the cell cytoplasm (large white squares show a magnified area of the O-IMV). Conventional OMVs are also visualized in images A and C (black arrows). OM: Outer Membrane; PM: Plasma membrane; PG: Peptidoglycan. Bars, 500 nm (A, C) and 250 nm (B).
Figure 5.
Quantification of O-IMVs on thin frozen foils from the total MVs using Cryo-TEM.
(A) Overview of a thin frozen foil obtained from A. baumannii. Single-layer vesicles are highly abundant, while double-layer O-IMVs can be observed in all the tracked fields, but much less often (white arrows). (B) Cryo-TEM images of thin frozen foils from the MVs of the three assayed strains. Both types of vesicles are observed, the single-layer OMVs and the new double-layer O-IMVs (white arrows). In A. baumannii O-IMVs, the presence of the putative intact peptidoglycan layer is also observed (PG). Bars 500 nm (A) and 200 nm (B).
Table 1.
Diameter mean values and diameter ranges expressed in nm from OMVs and O-IMVs for each analyzed strain.
Table 2.
DNA and ATP quantification in MVs.
Figure 6.
DNA gold immunolabeling on Lowicryl HM20 thin sections of HPF-FS isolated MVs from N. gonorrhoeae.
(A) TEM micrograph showing an O-IMV immunolabeled with a monoclonal IgM specific against dsDNA and a secondary goat anti-mouse antibody coupled to 12-nm colloidal gold. The gold mark is localized inside the inner layer that contains the electron-dense material, which confirms that the DNA is packaged within the new O-IMV. (B) TEM micrograph of MVs labeled only with the secondary antibody. (C) TEM micrograph of MVs from grids preincubated with 1 mg/ml DNase I and then immunolabeled with the anti-dsDNA IgM and a secondary antibody coupled to gold. Bars, 100 nm.
Figure 7.
Protein content from the N. gonorrhoeae-derived MVs.
(A) Distribution of proteins identified from the total MVs from N. gonorrhoeae based on their subcellular location. (B) Functional classification of the 51 proteins predicted to be localized in the cytoplasm and plasma membrane of N. gonorrhoeae cells.