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Interaction of Bacterial Membrane Vesicles with Specific Species and Their Potential for Delivery to Target Cells

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ABSTRACT

Membrane vesicles (MVs) are secreted from a wide range of microbial species and transfer their content to other cells. Although MVs play critical roles in bacterial communication, whether MVs selectively interact with bacterial cells in microbial communities is unclear. In this study, we investigated the specificity of the MV-cell interactions and evaluated the potential of MVs to target bacterial cells for delivery. MV association with bacterial cells was examined using a fluorescent membrane dye to label MVs. MVs derived from the enterobacterium Buttiauxella agrestis specifically interacted with cells of the parent strain but interacted less specifically with those of other genera tested in this study. Electron microscopic analyses showed that MVs were not only attached on B. agrestis cells but also fused to them. The interaction energy, which was characterized by hydrodynamic diameter and zeta potential based on the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, was significant low between MVs and cells in B. agrestis, compared to those between B. agrestis MVs and cells of other genera. Similar specific interaction was also occurred between B. agrestis MVs and cells of six other species belonging to Buttiauxella spp. B. agrestis harboring plasmid pBBR1MCS-1 secreted plasmid-containing MVs (p-MVs), and plasmid DNA in p-MVs was transferred to the same species. Moreover, antibiotic-associated MVs enabled effective killing of target species; the survival rate of B. agrestis was lower than those of Escherichia coli and Pseudomonas aeruginosa in the presence of gentamicin-associated MVs derived from B. agrestis. Altogether, we provide the evidence that MVs selectively interact with target bacterial cells and offer a new avenue for controlling specific bacterial species using bacterial MVs in microbial communities.

No MeSH data available.


TEM imaging. (A) Image of purified MVs derived from B. agrestis CUETM77-167. (B) Association of FM4-64-labeled MVs with cells. The white arrows indicate MVs, which have a high density due to FM4-64 labeling. (C) Association of FITC-labeled MVs with cells. Cell-associated MVs were detected by small gold particles (black arrows) through the FITC antibody. (D) Cells with no addition of MVs were reacted with FITC antibody. All bars indicate 100 nm.
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Figure 3: TEM imaging. (A) Image of purified MVs derived from B. agrestis CUETM77-167. (B) Association of FM4-64-labeled MVs with cells. The white arrows indicate MVs, which have a high density due to FM4-64 labeling. (C) Association of FITC-labeled MVs with cells. Cell-associated MVs were detected by small gold particles (black arrows) through the FITC antibody. (D) Cells with no addition of MVs were reacted with FITC antibody. All bars indicate 100 nm.

Mentions: We confirmed that the extract from the supernatant of B. agrestis culture from ultracentrifugation contained spherical MVs by TEM observation (Figure 3A). Next, we investigated if supplemented MVs interacted with bacterial cells using TEM observation. FM dyes have been used to determine the localization of supplemented vesicles because their photoactivation creates reactive oxygen species, which form an electron-dense precipitate in the vacuoles that is readily visualized under electron microscope (Harata et al., 2001). Then, FM4-64-labeled MVs were incubated with non-labeled cells of B. agrestis, and TEM analysis was conducted to observe the interaction of added MVs with B. agrestis cells. The result showed that MVs attached to the cellular surface were darkly stained (Figure 3B), suggesting that additional MVs were definitely associated with cells of B. agrestis. To further analyze MV-cell interaction, FITC-labeled MVs were incubated with non-labeled cells, and FITC was labeled by gold particles using a gold-conjugate antibody against FITC. The fusion of MVs onto bacterial cells was visualized, and MVs were labeled with gold particles (Figure 3C), suggesting that the MVs in the image were not those being released from cells but rather those being incorporated into cells. When MVs were not added to bacterial suspension before immunolabeling as the control, dense gold particles were not observed around cells (Figure 3D). Thus, the interaction of MVs with bacterial cells is not only the surface attachment but also the membranous fusion.


Interaction of Bacterial Membrane Vesicles with Specific Species and Their Potential for Delivery to Target Cells
TEM imaging. (A) Image of purified MVs derived from B. agrestis CUETM77-167. (B) Association of FM4-64-labeled MVs with cells. The white arrows indicate MVs, which have a high density due to FM4-64 labeling. (C) Association of FITC-labeled MVs with cells. Cell-associated MVs were detected by small gold particles (black arrows) through the FITC antibody. (D) Cells with no addition of MVs were reacted with FITC antibody. All bars indicate 100 nm.
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Figure 3: TEM imaging. (A) Image of purified MVs derived from B. agrestis CUETM77-167. (B) Association of FM4-64-labeled MVs with cells. The white arrows indicate MVs, which have a high density due to FM4-64 labeling. (C) Association of FITC-labeled MVs with cells. Cell-associated MVs were detected by small gold particles (black arrows) through the FITC antibody. (D) Cells with no addition of MVs were reacted with FITC antibody. All bars indicate 100 nm.
Mentions: We confirmed that the extract from the supernatant of B. agrestis culture from ultracentrifugation contained spherical MVs by TEM observation (Figure 3A). Next, we investigated if supplemented MVs interacted with bacterial cells using TEM observation. FM dyes have been used to determine the localization of supplemented vesicles because their photoactivation creates reactive oxygen species, which form an electron-dense precipitate in the vacuoles that is readily visualized under electron microscope (Harata et al., 2001). Then, FM4-64-labeled MVs were incubated with non-labeled cells of B. agrestis, and TEM analysis was conducted to observe the interaction of added MVs with B. agrestis cells. The result showed that MVs attached to the cellular surface were darkly stained (Figure 3B), suggesting that additional MVs were definitely associated with cells of B. agrestis. To further analyze MV-cell interaction, FITC-labeled MVs were incubated with non-labeled cells, and FITC was labeled by gold particles using a gold-conjugate antibody against FITC. The fusion of MVs onto bacterial cells was visualized, and MVs were labeled with gold particles (Figure 3C), suggesting that the MVs in the image were not those being released from cells but rather those being incorporated into cells. When MVs were not added to bacterial suspension before immunolabeling as the control, dense gold particles were not observed around cells (Figure 3D). Thus, the interaction of MVs with bacterial cells is not only the surface attachment but also the membranous fusion.

View Article: PubMed Central - PubMed

ABSTRACT

Membrane vesicles (MVs) are secreted from a wide range of microbial species and transfer their content to other cells. Although MVs play critical roles in bacterial communication, whether MVs selectively interact with bacterial cells in microbial communities is unclear. In this study, we investigated the specificity of the MV-cell interactions and evaluated the potential of MVs to target bacterial cells for delivery. MV association with bacterial cells was examined using a fluorescent membrane dye to label MVs. MVs derived from the enterobacterium Buttiauxella agrestis specifically interacted with cells of the parent strain but interacted less specifically with those of other genera tested in this study. Electron microscopic analyses showed that MVs were not only attached on B. agrestis cells but also fused to them. The interaction energy, which was characterized by hydrodynamic diameter and zeta potential based on the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, was significant low between MVs and cells in B. agrestis, compared to those between B. agrestis MVs and cells of other genera. Similar specific interaction was also occurred between B. agrestis MVs and cells of six other species belonging to Buttiauxella spp. B. agrestis harboring plasmid pBBR1MCS-1 secreted plasmid-containing MVs (p-MVs), and plasmid DNA in p-MVs was transferred to the same species. Moreover, antibiotic-associated MVs enabled effective killing of target species; the survival rate of B. agrestis was lower than those of Escherichia coli and Pseudomonas aeruginosa in the presence of gentamicin-associated MVs derived from B. agrestis. Altogether, we provide the evidence that MVs selectively interact with target bacterial cells and offer a new avenue for controlling specific bacterial species using bacterial MVs in microbial communities.

No MeSH data available.