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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.


Selective interactions between cells and MVs are conserved in Buttiauxella spp. (A) The phylogenetic relationship between Buttiauxella spp. and E. coli. Numbers on the branching points are the percentages of bootstrap values with 1000 replicates. (B) Association of MVs derived from B. agrestis CUETM77-167 with bacterial cells. FM4-64-labeled MVs (20 μg/mL of phospholipids) were incubated with bacterial cells for 30 min at 30°C. MVs associated with cells were identified by RFUs of FM4-64 normalized to the cellular protein concentration (mg/mL). The data are shown as the mean ± standard deviation from three replicates. (C) Primary maximum energy between each cell and B. agrestis MVs. The values were calculated from the zeta potential and particle size based on the DLVO theory. (D) Relationship between the MV association with cells and primary maximum energy. Blue plots show Buttiauxella strains (from B,C), and red plots show other bacterial strains (from Figures 1B, 4C). The coefficient of determination R2 is 0.59.
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Figure 5: Selective interactions between cells and MVs are conserved in Buttiauxella spp. (A) The phylogenetic relationship between Buttiauxella spp. and E. coli. Numbers on the branching points are the percentages of bootstrap values with 1000 replicates. (B) Association of MVs derived from B. agrestis CUETM77-167 with bacterial cells. FM4-64-labeled MVs (20 μg/mL of phospholipids) were incubated with bacterial cells for 30 min at 30°C. MVs associated with cells were identified by RFUs of FM4-64 normalized to the cellular protein concentration (mg/mL). The data are shown as the mean ± standard deviation from three replicates. (C) Primary maximum energy between each cell and B. agrestis MVs. The values were calculated from the zeta potential and particle size based on the DLVO theory. (D) Relationship between the MV association with cells and primary maximum energy. Blue plots show Buttiauxella strains (from B,C), and red plots show other bacterial strains (from Figures 1B, 4C). The coefficient of determination R2 is 0.59.

Mentions: Although a specific interaction of MVs with the same species was observed in B. agrestis, it was unknown whether this characteristic was confined to this bacterium. We prepared 6 other Buttiauxella strains (B. brennerae S1/6-571, B. ferragutiae CDC1180-81, B. noackiae NSW11, B. izardii S3/2-161, B. warmboldiae NSW326, and B. gaviniae S1/1-984), and the phylogenetic relationship among them and E. coli is shown in Figure 5A. Interestingly, the MVs derived from B. agrestis CUETM77-167 showed a much higher association with cells of Buttiauxella spp. than with E. coli (Figure 5B), suggesting that the specific interaction of MVs with cells is conserved in Buttiauxella spp. To determine whether the high interaction between MVs derived from CUETM77-167 and Buttiauxella strains is due to the physicochemical interaction energy, the zeta potentials and hydrodynamic diameters of each cell were examined. The calculated interaction energy based on the DLVO theory showed that the primary maximum energies of several Buttiauxella cells with MVs derived from CUETM77-167 were significantly lower than that for E. coli (Figure 5C and Supplementary Figure S5). We next evaluated the relationship between the primary maximum energies calculated by DLVO theory and the association values of MVs with bacterial cells. Figure 5D shows that plots of Buttiauxella strains are localized at different positions from those of other strains. Statistical analyses indicated significant differences between Buttiauxella strains and other strains in both association with B. agrestis MVs and primary maximum energies (Supplementary Figure S6), suggesting that interaction energy based on the DLVO theory is one factor explaining why MVs specifically interact with bacterial cells of Buttiauxella spp. However, the correlation between the two parameters is not linear in Figure 5D (the coefficient of determination R2 is 0.59), and other factors besides low interaction energy based on the DLVO theory may affect the specific interaction of MVs in Buttiauxella strains. When MVs and/or cells were treated with proteinase K, the association between MVs and cells in B. agrestis was decreased more than 50% (Supplementary Figure S7), suggesting that proteins localized on the surface of cells and MVs also affect the MV-cell interaction in B. agrestis.


Interaction of Bacterial Membrane Vesicles with Specific Species and Their Potential for Delivery to Target Cells
Selective interactions between cells and MVs are conserved in Buttiauxella spp. (A) The phylogenetic relationship between Buttiauxella spp. and E. coli. Numbers on the branching points are the percentages of bootstrap values with 1000 replicates. (B) Association of MVs derived from B. agrestis CUETM77-167 with bacterial cells. FM4-64-labeled MVs (20 μg/mL of phospholipids) were incubated with bacterial cells for 30 min at 30°C. MVs associated with cells were identified by RFUs of FM4-64 normalized to the cellular protein concentration (mg/mL). The data are shown as the mean ± standard deviation from three replicates. (C) Primary maximum energy between each cell and B. agrestis MVs. The values were calculated from the zeta potential and particle size based on the DLVO theory. (D) Relationship between the MV association with cells and primary maximum energy. Blue plots show Buttiauxella strains (from B,C), and red plots show other bacterial strains (from Figures 1B, 4C). The coefficient of determination R2 is 0.59.
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Figure 5: Selective interactions between cells and MVs are conserved in Buttiauxella spp. (A) The phylogenetic relationship between Buttiauxella spp. and E. coli. Numbers on the branching points are the percentages of bootstrap values with 1000 replicates. (B) Association of MVs derived from B. agrestis CUETM77-167 with bacterial cells. FM4-64-labeled MVs (20 μg/mL of phospholipids) were incubated with bacterial cells for 30 min at 30°C. MVs associated with cells were identified by RFUs of FM4-64 normalized to the cellular protein concentration (mg/mL). The data are shown as the mean ± standard deviation from three replicates. (C) Primary maximum energy between each cell and B. agrestis MVs. The values were calculated from the zeta potential and particle size based on the DLVO theory. (D) Relationship between the MV association with cells and primary maximum energy. Blue plots show Buttiauxella strains (from B,C), and red plots show other bacterial strains (from Figures 1B, 4C). The coefficient of determination R2 is 0.59.
Mentions: Although a specific interaction of MVs with the same species was observed in B. agrestis, it was unknown whether this characteristic was confined to this bacterium. We prepared 6 other Buttiauxella strains (B. brennerae S1/6-571, B. ferragutiae CDC1180-81, B. noackiae NSW11, B. izardii S3/2-161, B. warmboldiae NSW326, and B. gaviniae S1/1-984), and the phylogenetic relationship among them and E. coli is shown in Figure 5A. Interestingly, the MVs derived from B. agrestis CUETM77-167 showed a much higher association with cells of Buttiauxella spp. than with E. coli (Figure 5B), suggesting that the specific interaction of MVs with cells is conserved in Buttiauxella spp. To determine whether the high interaction between MVs derived from CUETM77-167 and Buttiauxella strains is due to the physicochemical interaction energy, the zeta potentials and hydrodynamic diameters of each cell were examined. The calculated interaction energy based on the DLVO theory showed that the primary maximum energies of several Buttiauxella cells with MVs derived from CUETM77-167 were significantly lower than that for E. coli (Figure 5C and Supplementary Figure S5). We next evaluated the relationship between the primary maximum energies calculated by DLVO theory and the association values of MVs with bacterial cells. Figure 5D shows that plots of Buttiauxella strains are localized at different positions from those of other strains. Statistical analyses indicated significant differences between Buttiauxella strains and other strains in both association with B. agrestis MVs and primary maximum energies (Supplementary Figure S6), suggesting that interaction energy based on the DLVO theory is one factor explaining why MVs specifically interact with bacterial cells of Buttiauxella spp. However, the correlation between the two parameters is not linear in Figure 5D (the coefficient of determination R2 is 0.59), and other factors besides low interaction energy based on the DLVO theory may affect the specific interaction of MVs in Buttiauxella strains. When MVs and/or cells were treated with proteinase K, the association between MVs and cells in B. agrestis was decreased more than 50% (Supplementary Figure S7), suggesting that proteins localized on the surface of cells and MVs also affect the MV-cell interaction in B. agrestis.

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.