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

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.


Transfer of gentamicin through MVs derived from B. agrestis. (A–C) Survival rate of B. agrestis CUETM77-167 (A), E. coli MG1655 (B) or P. aeruginosa PAO1 (C). Each bacterial cell was incubated with normal MVs (n-MVs: green), gentamicin-harboring MVs (g-MVs: red) and homogenized g-MVs (blue). The data are shown as the mean ± standard deviation from three replicates. (D) Survival rate of bacterial cells in the mixture of CUETM77-167 (red) and E. coli DH5α (blue). The samples were incubated with n-MVs (squares), g-MVs (triangles), and homogenized g-MVs (circles). The data are shown as the mean ± standard deviation from three replicates.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383704&req=5

Figure 7: Transfer of gentamicin through MVs derived from B. agrestis. (A–C) Survival rate of B. agrestis CUETM77-167 (A), E. coli MG1655 (B) or P. aeruginosa PAO1 (C). Each bacterial cell was incubated with normal MVs (n-MVs: green), gentamicin-harboring MVs (g-MVs: red) and homogenized g-MVs (blue). The data are shown as the mean ± standard deviation from three replicates. (D) Survival rate of bacterial cells in the mixture of CUETM77-167 (red) and E. coli DH5α (blue). The samples were incubated with n-MVs (squares), g-MVs (triangles), and homogenized g-MVs (circles). The data are shown as the mean ± standard deviation from three replicates.

Mentions: We expected that the specific interaction of MVs with bacterial cells could be used for the selective control of bacterial cells. Exposure to the antibiotic gentamicin has been shown to increase MV formation, and gentamicin was retained within the MVs (Kadurugamuwa and Beveridge, 1995; Fulsundar et al., 2014). We evaluated the potential of MVs as tools for the delivery of gentamicin to specific bacterial cells. B. agrestis was grown to the stationary phase, and gentamicin was added to the culture at a final concentration of 32 μg/mL (four times the MIC). MVs were extracted, and gentamicin was concentrated in MVs (146 μg/mL) based on the calculated gentamicin concentration and the number of MVs. We prepared gentamicin-associated MVs (g-MVs) and homogenized g-MVs, in which the gentamicin concentration was likely the same as that in the sample of g-MVs. As a negative control, MVs derived from B. agrestis not associated with gentamicin (n-MVs) were also prepared. These MVs were incubated with cells of B. agrestis, E. coli, and P. aeruginosa, and the survival rates were examined. The killing effect of g-MVs on B. agrestis was much greater than that on E. coli and P. aeruginosa, while homogenized g-MVs had a high killing effect on all of the bacteria that were tested in this experiment (Figures 7A–C). Under these experimental conditions, the gentamicin concentration of each sample ranged from 1.3 to 2.3 μg/mL, and this concentration was lower than the MIC of each strain. Subsequently, to determine whether selective antibiotic delivery via MVs occurred in the microbial complexes, the effect of g-MVs in the mixed samples containing B. agrestis and E. coli cells was examined. The results showed that the killing effect of g-MVs on B. agrestis was higher than that on E. coli, even in mixed samples (Figure 7D). These data indicate that the specific interaction of B. agrestis MVs is useful for killing target species in heterogeneous samples.


Interaction of Bacterial Membrane Vesicles with Specific Species and Their Potential for Delivery to Target Cells
Transfer of gentamicin through MVs derived from B. agrestis. (A–C) Survival rate of B. agrestis CUETM77-167 (A), E. coli MG1655 (B) or P. aeruginosa PAO1 (C). Each bacterial cell was incubated with normal MVs (n-MVs: green), gentamicin-harboring MVs (g-MVs: red) and homogenized g-MVs (blue). The data are shown as the mean ± standard deviation from three replicates. (D) Survival rate of bacterial cells in the mixture of CUETM77-167 (red) and E. coli DH5α (blue). The samples were incubated with n-MVs (squares), g-MVs (triangles), and homogenized g-MVs (circles). The data are shown as the mean ± standard deviation from three replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383704&req=5

Figure 7: Transfer of gentamicin through MVs derived from B. agrestis. (A–C) Survival rate of B. agrestis CUETM77-167 (A), E. coli MG1655 (B) or P. aeruginosa PAO1 (C). Each bacterial cell was incubated with normal MVs (n-MVs: green), gentamicin-harboring MVs (g-MVs: red) and homogenized g-MVs (blue). The data are shown as the mean ± standard deviation from three replicates. (D) Survival rate of bacterial cells in the mixture of CUETM77-167 (red) and E. coli DH5α (blue). The samples were incubated with n-MVs (squares), g-MVs (triangles), and homogenized g-MVs (circles). The data are shown as the mean ± standard deviation from three replicates.
Mentions: We expected that the specific interaction of MVs with bacterial cells could be used for the selective control of bacterial cells. Exposure to the antibiotic gentamicin has been shown to increase MV formation, and gentamicin was retained within the MVs (Kadurugamuwa and Beveridge, 1995; Fulsundar et al., 2014). We evaluated the potential of MVs as tools for the delivery of gentamicin to specific bacterial cells. B. agrestis was grown to the stationary phase, and gentamicin was added to the culture at a final concentration of 32 μg/mL (four times the MIC). MVs were extracted, and gentamicin was concentrated in MVs (146 μg/mL) based on the calculated gentamicin concentration and the number of MVs. We prepared gentamicin-associated MVs (g-MVs) and homogenized g-MVs, in which the gentamicin concentration was likely the same as that in the sample of g-MVs. As a negative control, MVs derived from B. agrestis not associated with gentamicin (n-MVs) were also prepared. These MVs were incubated with cells of B. agrestis, E. coli, and P. aeruginosa, and the survival rates were examined. The killing effect of g-MVs on B. agrestis was much greater than that on E. coli and P. aeruginosa, while homogenized g-MVs had a high killing effect on all of the bacteria that were tested in this experiment (Figures 7A–C). Under these experimental conditions, the gentamicin concentration of each sample ranged from 1.3 to 2.3 μg/mL, and this concentration was lower than the MIC of each strain. Subsequently, to determine whether selective antibiotic delivery via MVs occurred in the microbial complexes, the effect of g-MVs in the mixed samples containing B. agrestis and E. coli cells was examined. The results showed that the killing effect of g-MVs on B. agrestis was higher than that on E. coli, even in mixed samples (Figure 7D). These data indicate that the specific interaction of B. agrestis MVs is useful for killing target species in heterogeneous samples.

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.