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Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN - PLoS ONE (2015)

Bottom Line: We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells.By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined.Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, S-90187, Sweden; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, S-90187, Sweden.

ABSTRACT

Background: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV.

Methodology/principal findings: In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37.

Conclusion/significance: Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE, immunoblot analyses, Nanoparticle tracking analysis and electron microscopic analyses of OMV samples from the wild type strain C6706 and the prtV mutant.(A) SDS-PAGE and Coomassie blue staining of OMVs samples from V. cholerae wild type C6706 (lane 1) and its derivative prtV mutant (lane 2). (B) Immunoblot analysis of PrtV protein in the OMV samples from the wild type strain C6706 (upper panel, lane 1) and the prtV mutant (upper panel, lane 2) using anti-PrtV polyclonal antiserum. Immunoblot analysis of OMV samples using anti-OmpU antiserum as a OMV marker (middle panel) and anti-Crp polyclonal antiserum as a cytoplasmic protein marker (lower panel). (C) Nanoparticle tracking analysis measurement of OMVs isolated from the wild type V. cholerae strain C6706 showing the sizes and total concentration of OMVs. (D) Nanoparticle tracking analysis measurement of OMVs isolated from the ΔprtV mutant showing the sizes and total concentration of OMVs. (E) Electron microscopy of OMVs from the wild type V. cholerae strain C6706 (a) and the prtV mutant (b). Immunogold labeling of OMVs from V. cholerae wild type strain C6706 (c) and the prtV mutant (d). White arrow points to gold particles associated with OMVs. Bars; 150 nm.
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pone.0134098.g003: SDS-PAGE, immunoblot analyses, Nanoparticle tracking analysis and electron microscopic analyses of OMV samples from the wild type strain C6706 and the prtV mutant.(A) SDS-PAGE and Coomassie blue staining of OMVs samples from V. cholerae wild type C6706 (lane 1) and its derivative prtV mutant (lane 2). (B) Immunoblot analysis of PrtV protein in the OMV samples from the wild type strain C6706 (upper panel, lane 1) and the prtV mutant (upper panel, lane 2) using anti-PrtV polyclonal antiserum. Immunoblot analysis of OMV samples using anti-OmpU antiserum as a OMV marker (middle panel) and anti-Crp polyclonal antiserum as a cytoplasmic protein marker (lower panel). (C) Nanoparticle tracking analysis measurement of OMVs isolated from the wild type V. cholerae strain C6706 showing the sizes and total concentration of OMVs. (D) Nanoparticle tracking analysis measurement of OMVs isolated from the ΔprtV mutant showing the sizes and total concentration of OMVs. (E) Electron microscopy of OMVs from the wild type V. cholerae strain C6706 (a) and the prtV mutant (b). Immunogold labeling of OMVs from V. cholerae wild type strain C6706 (c) and the prtV mutant (d). White arrow points to gold particles associated with OMVs. Bars; 150 nm.

Mentions: To further examine the OMVs from the wild type and prtV mutant strains, gradient fractions number 8 from C6706 and its prtV mutant were analysed. As shown by SDS-PAGE and Coomassie blue staining (Fig 3A), the OMV fraction from these two strains exhibited almost identical protein profiles. Immunoblotting confirmed the presence of PrtV in the C6706 OMV fraction only (Fig 3B, upper panel). As was observed in Fig 1C, we detected two PrtV bands at 81 kDa and 37 kDa, respectively. The 37 kDa might be an autoproteolytic form of PrtV protein in the OMVs. As judged by the protein profiles (Fig 3A) and the intensity of the OmpU band in the OMV samples from the wild type C6707 and the prtV mutant (Fig 3B, middle panel), the amount of OMVs released from the wild type and the prtV mutant was very similar. The total protein content of each OMV sample was measured using the Bicinchoninic Acid (BCA) assay kit as described in the materials and methods. It showed that OMVs from the wild type and ΔprtV mutant bacteria contain 1,090 μg/ml and 1,270 μg/ml protein, respectively. We used nanoparticle tracking analysis (NTA), a new method for direct, real-time visualization of nanoparticles in liquids [32]. In this system, OMVs can be observed by light scattering using a light microscope. A video was taken, and the NTA software can track the brownian movement of individual OMVs and calculate the size and concentration of OMVs. The amount of OMV particles measured by nanoparticle tracking analysis using the NanoSight equipment are shown in Fig 3C and 3D, the OMV samples from the wild type C6706 and ΔprtV mutant contained 7.5 x 1012/ml (Fig 3C) and 8.5 x 1012/ml OMV-particles (Fig 3D) respectively. The size distribution of OMVs isolated from both the wild type and ΔprtV mutant was in the 50–250 μm diameter range with the majority of the OMV particles at 105 μm from both the wild type and the ΔprtV mutant V. cholerae (Fig 3C and 3D). Interestingly, an extra peak representing 155 μm diameter sized OMVs was observed in the wild type OMV sample (Fig 3C). It could be considered that the soluble form of PrtV might form particles showing up as 155 nm on the nanoparticle tracking analysis since this method presumably cannot distinguish between different types of particles. The morphology of OMVs was examined by transmission electron microscopy, which also revealed similar sizes and morphology of OMVs from the wild type and the prtV mutant (Fig 3E, panels a and b). To test for possible contamination from lysed bacterial cells in these gradient fractions, immunoblotting was also carried out using antiserum against the cytoplasmic cAMP receptor protein (Crp). As this revealed no Crp reactive bands (Fig 3B, lower panel), we concluded that there was no detectable cytoplasmic contamination in these samples. In order to visualize the association of PrtV with OMVs, we carried out electron microscopy analysis and immunogold labeling using PrtV polyclonal antiserum. OMV-associated several gold particles were observed in the wild type strain, whereas no gold particles were associated with OMVs isolated from the prtV mutant (Fig 3E, panels c and d). Taken together, our results strongly support the idea that the PrtV protein is associated with OMVs released from V. cholerae.


Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN - PLoS ONE (2015)

SDS-PAGE, immunoblot analyses, Nanoparticle tracking analysis and electron microscopic analyses of OMV samples from the wild type strain C6706 and the prtV mutant.(A) SDS-PAGE and Coomassie blue staining of OMVs samples from V. cholerae wild type C6706 (lane 1) and its derivative prtV mutant (lane 2). (B) Immunoblot analysis of PrtV protein in the OMV samples from the wild type strain C6706 (upper panel, lane 1) and the prtV mutant (upper panel, lane 2) using anti-PrtV polyclonal antiserum. Immunoblot analysis of OMV samples using anti-OmpU antiserum as a OMV marker (middle panel) and anti-Crp polyclonal antiserum as a cytoplasmic protein marker (lower panel). (C) Nanoparticle tracking analysis measurement of OMVs isolated from the wild type V. cholerae strain C6706 showing the sizes and total concentration of OMVs. (D) Nanoparticle tracking analysis measurement of OMVs isolated from the ΔprtV mutant showing the sizes and total concentration of OMVs. (E) Electron microscopy of OMVs from the wild type V. cholerae strain C6706 (a) and the prtV mutant (b). Immunogold labeling of OMVs from V. cholerae wild type strain C6706 (c) and the prtV mutant (d). White arrow points to gold particles associated with OMVs. Bars; 150 nm.
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Related In: Results  -  Collection

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pone.0134098.g003: SDS-PAGE, immunoblot analyses, Nanoparticle tracking analysis and electron microscopic analyses of OMV samples from the wild type strain C6706 and the prtV mutant.(A) SDS-PAGE and Coomassie blue staining of OMVs samples from V. cholerae wild type C6706 (lane 1) and its derivative prtV mutant (lane 2). (B) Immunoblot analysis of PrtV protein in the OMV samples from the wild type strain C6706 (upper panel, lane 1) and the prtV mutant (upper panel, lane 2) using anti-PrtV polyclonal antiserum. Immunoblot analysis of OMV samples using anti-OmpU antiserum as a OMV marker (middle panel) and anti-Crp polyclonal antiserum as a cytoplasmic protein marker (lower panel). (C) Nanoparticle tracking analysis measurement of OMVs isolated from the wild type V. cholerae strain C6706 showing the sizes and total concentration of OMVs. (D) Nanoparticle tracking analysis measurement of OMVs isolated from the ΔprtV mutant showing the sizes and total concentration of OMVs. (E) Electron microscopy of OMVs from the wild type V. cholerae strain C6706 (a) and the prtV mutant (b). Immunogold labeling of OMVs from V. cholerae wild type strain C6706 (c) and the prtV mutant (d). White arrow points to gold particles associated with OMVs. Bars; 150 nm.
Mentions: To further examine the OMVs from the wild type and prtV mutant strains, gradient fractions number 8 from C6706 and its prtV mutant were analysed. As shown by SDS-PAGE and Coomassie blue staining (Fig 3A), the OMV fraction from these two strains exhibited almost identical protein profiles. Immunoblotting confirmed the presence of PrtV in the C6706 OMV fraction only (Fig 3B, upper panel). As was observed in Fig 1C, we detected two PrtV bands at 81 kDa and 37 kDa, respectively. The 37 kDa might be an autoproteolytic form of PrtV protein in the OMVs. As judged by the protein profiles (Fig 3A) and the intensity of the OmpU band in the OMV samples from the wild type C6707 and the prtV mutant (Fig 3B, middle panel), the amount of OMVs released from the wild type and the prtV mutant was very similar. The total protein content of each OMV sample was measured using the Bicinchoninic Acid (BCA) assay kit as described in the materials and methods. It showed that OMVs from the wild type and ΔprtV mutant bacteria contain 1,090 μg/ml and 1,270 μg/ml protein, respectively. We used nanoparticle tracking analysis (NTA), a new method for direct, real-time visualization of nanoparticles in liquids [32]. In this system, OMVs can be observed by light scattering using a light microscope. A video was taken, and the NTA software can track the brownian movement of individual OMVs and calculate the size and concentration of OMVs. The amount of OMV particles measured by nanoparticle tracking analysis using the NanoSight equipment are shown in Fig 3C and 3D, the OMV samples from the wild type C6706 and ΔprtV mutant contained 7.5 x 1012/ml (Fig 3C) and 8.5 x 1012/ml OMV-particles (Fig 3D) respectively. The size distribution of OMVs isolated from both the wild type and ΔprtV mutant was in the 50–250 μm diameter range with the majority of the OMV particles at 105 μm from both the wild type and the ΔprtV mutant V. cholerae (Fig 3C and 3D). Interestingly, an extra peak representing 155 μm diameter sized OMVs was observed in the wild type OMV sample (Fig 3C). It could be considered that the soluble form of PrtV might form particles showing up as 155 nm on the nanoparticle tracking analysis since this method presumably cannot distinguish between different types of particles. The morphology of OMVs was examined by transmission electron microscopy, which also revealed similar sizes and morphology of OMVs from the wild type and the prtV mutant (Fig 3E, panels a and b). To test for possible contamination from lysed bacterial cells in these gradient fractions, immunoblotting was also carried out using antiserum against the cytoplasmic cAMP receptor protein (Crp). As this revealed no Crp reactive bands (Fig 3B, lower panel), we concluded that there was no detectable cytoplasmic contamination in these samples. In order to visualize the association of PrtV with OMVs, we carried out electron microscopy analysis and immunogold labeling using PrtV polyclonal antiserum. OMV-associated several gold particles were observed in the wild type strain, whereas no gold particles were associated with OMVs isolated from the prtV mutant (Fig 3E, panels c and d). Taken together, our results strongly support the idea that the PrtV protein is associated with OMVs released from V. cholerae.

Bottom Line: We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells.By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined.Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, S-90187, Sweden; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, S-90187, Sweden.

ABSTRACT

Background: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV.

Methodology/principal findings: In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37.

Conclusion/significance: Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

No MeSH data available.


Related in: MedlinePlus