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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.


Plasmid transfer through MVs derived from B. agrestis. MVs were extracted from the culture of CUETM77-167 (open) or CUETM77-167/pBBR1-MCS (closed). DNase-treated (red triangles) or non-treated (blue squares) MVs (20 μg/mL of phospholipids) were incubated with CUETM77-167 at 30°C. Transformants were calculated by counting the CFUs on agar medium containing chloramphenicol. The data are shown as the mean ± standard deviation from three replicates.
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Figure 6: Plasmid transfer through MVs derived from B. agrestis. MVs were extracted from the culture of CUETM77-167 (open) or CUETM77-167/pBBR1-MCS (closed). DNase-treated (red triangles) or non-treated (blue squares) MVs (20 μg/mL of phospholipids) were incubated with CUETM77-167 at 30°C. Transformants were calculated by counting the CFUs on agar medium containing chloramphenicol. The data are shown as the mean ± standard deviation from three replicates.

Mentions: To determine whether the interaction of MVs with bacterial cells contributes to the delivery of the MV contents to bacterial cells, we evaluated vesicle-mediated plasmid DNA transfer. B. agrestis harboring pBBR1MCS-1 was grown in TSB medium to the stationary phase, and plasmid-containing MVs (p-MVs) were extracted from the supernatant. An examination of the pBBR1MCS-1 concentration by quantitative PCR showed 3.11 × 106 and 2.27 × 106 copies/mL in the supernatant before and after the removal of MVs through ultracentrifugation, respectively, suggesting that at least approximately one-third of the plasmid localized in the extracellular milieu was associated with p-MVs in the B. agrestis supernatant. The external DNA surrounding p-MVs was degraded by DNase I treatment, and the plasmid concentration in the p-MVs was calculated by counting the number of MVs using nano tracking analysis. The results showed that p-MVs contain 1.03 × 109 copies/mL of pBBR1MCS-1, indicating that p-MVs maintain a high concentration of plasmid and that the DNA in p-MVs was stable against DNase I treatment. When B. agrestis cells (approximately 1.0 × 103 cells/mL) were treated with an excessive amount of p-MVs, more than 30% B. agrestis transformants were obtained after 3 h of incubation with DNase I-treated p-MVs and non-treated p-MVs (Figure 6), suggesting that the plasmid contained in MVs was transferred to bacterial cells. When naked plasmid DNA instead of p-MVs was added to directly bacterial cell suspension in this experiment (data not shown), indicating that natural transformation was not occurred in the condition. Notably, other methods for DNA transformation into this strain have not yet been established in our experiments, suggesting that DNA transfer via p-MVs is a useful tool to obtain transformants in this strain.


Interaction of Bacterial Membrane Vesicles with Specific Species and Their Potential for Delivery to Target Cells
Plasmid transfer through MVs derived from B. agrestis. MVs were extracted from the culture of CUETM77-167 (open) or CUETM77-167/pBBR1-MCS (closed). DNase-treated (red triangles) or non-treated (blue squares) MVs (20 μg/mL of phospholipids) were incubated with CUETM77-167 at 30°C. Transformants were calculated by counting the CFUs on agar medium containing chloramphenicol. The data are shown as the mean ± standard deviation from three replicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383704&req=5

Figure 6: Plasmid transfer through MVs derived from B. agrestis. MVs were extracted from the culture of CUETM77-167 (open) or CUETM77-167/pBBR1-MCS (closed). DNase-treated (red triangles) or non-treated (blue squares) MVs (20 μg/mL of phospholipids) were incubated with CUETM77-167 at 30°C. Transformants were calculated by counting the CFUs on agar medium containing chloramphenicol. The data are shown as the mean ± standard deviation from three replicates.
Mentions: To determine whether the interaction of MVs with bacterial cells contributes to the delivery of the MV contents to bacterial cells, we evaluated vesicle-mediated plasmid DNA transfer. B. agrestis harboring pBBR1MCS-1 was grown in TSB medium to the stationary phase, and plasmid-containing MVs (p-MVs) were extracted from the supernatant. An examination of the pBBR1MCS-1 concentration by quantitative PCR showed 3.11 × 106 and 2.27 × 106 copies/mL in the supernatant before and after the removal of MVs through ultracentrifugation, respectively, suggesting that at least approximately one-third of the plasmid localized in the extracellular milieu was associated with p-MVs in the B. agrestis supernatant. The external DNA surrounding p-MVs was degraded by DNase I treatment, and the plasmid concentration in the p-MVs was calculated by counting the number of MVs using nano tracking analysis. The results showed that p-MVs contain 1.03 × 109 copies/mL of pBBR1MCS-1, indicating that p-MVs maintain a high concentration of plasmid and that the DNA in p-MVs was stable against DNase I treatment. When B. agrestis cells (approximately 1.0 × 103 cells/mL) were treated with an excessive amount of p-MVs, more than 30% B. agrestis transformants were obtained after 3 h of incubation with DNase I-treated p-MVs and non-treated p-MVs (Figure 6), suggesting that the plasmid contained in MVs was transferred to bacterial cells. When naked plasmid DNA instead of p-MVs was added to directly bacterial cell suspension in this experiment (data not shown), indicating that natural transformation was not occurred in the condition. Notably, other methods for DNA transformation into this strain have not yet been established in our experiments, suggesting that DNA transfer via p-MVs is a useful tool to obtain transformants in this strain.

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.