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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

Presence of PrtV in OMVs from V. cholerae strain C6706.To detect the PrtV and the OmpU proteins in the density gradient fractions, immunoblot analyses were performed using polyclonal anti-PrtV and anti-OmpU antisera, respectively. (A) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the wild type V. cholerae strain C6706. (B) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the prtV mutant. (C) Immunobot detection of PrtV in the whole cell lysate (lane 1), culture supernatant before ultracentrifugation (lane 2), supernatant after the removal of OMVs (lane 3), and OMV sample (lane 4).
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pone.0134098.g002: Presence of PrtV in OMVs from V. cholerae strain C6706.To detect the PrtV and the OmpU proteins in the density gradient fractions, immunoblot analyses were performed using polyclonal anti-PrtV and anti-OmpU antisera, respectively. (A) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the wild type V. cholerae strain C6706. (B) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the prtV mutant. (C) Immunobot detection of PrtV in the whole cell lysate (lane 1), culture supernatant before ultracentrifugation (lane 2), supernatant after the removal of OMVs (lane 3), and OMV sample (lane 4).

Mentions: To confirm the secretion of PrtV in association with OMVs, vesicles from strain C6706 were isolated and purified using an Optiprep density gradient centrifugation, as described in Materials and Methods. Analysis of the Optiprep fractions by immunoblotting using anti-PrtV polyclonal antibody revealed the presence of PrtV protein in fractions 7–10 (Fig 2A, upper panel). The presence of outer membrane protein in these fractions was confirmed by immunoblotting using anti-OmpU polyclonal antibody (Fig 2A, lower panel). As a control experiment, we analysed OMVs from the prtV mutant derivative of C6706 using the same approach. The absence of PrtV in the density gradient fractions was confirmed by immunoblotting, whereas OmpU was detected in fractions 5–10 (Fig 2B, upper and lower panels, respectively). Based on these observations we concluded that PrtV was secreted in association with vesicles. To estimate what percentage of the secreted PrtV was with associated with OMVs (i) total secreted PrtV in the cell-free culture supernatants (before OMV isolation); (ii) soluble PrtV (supernatant after separation of the OMVs by ultracentrifugation); and (iii) OMV-associated PrtV (purified vesicle sample) were examined for three independent cultures of the strain C6706 by immunoblotting. The immunoblot analysis of a representative set of samples is shown in Fig 2C. For each culture the amount of total secreted PrtV was given arbitrarily the value of 100. The results, given as a percentage, indicated that most of the secreted PrtV was associated with vesicles (70 ± 5%), whereas a smaller fraction was present in a free soluble form in the supernatant (30 ± 5%).


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)

Presence of PrtV in OMVs from V. cholerae strain C6706.To detect the PrtV and the OmpU proteins in the density gradient fractions, immunoblot analyses were performed using polyclonal anti-PrtV and anti-OmpU antisera, respectively. (A) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the wild type V. cholerae strain C6706. (B) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the prtV mutant. (C) Immunobot detection of PrtV in the whole cell lysate (lane 1), culture supernatant before ultracentrifugation (lane 2), supernatant after the removal of OMVs (lane 3), and OMV sample (lane 4).
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Related In: Results  -  Collection

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pone.0134098.g002: Presence of PrtV in OMVs from V. cholerae strain C6706.To detect the PrtV and the OmpU proteins in the density gradient fractions, immunoblot analyses were performed using polyclonal anti-PrtV and anti-OmpU antisera, respectively. (A) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the wild type V. cholerae strain C6706. (B) Immunoblot detection of PrtV (upper panel) and OmpU (lower panel) in density gradient fractions of OMVs from the prtV mutant. (C) Immunobot detection of PrtV in the whole cell lysate (lane 1), culture supernatant before ultracentrifugation (lane 2), supernatant after the removal of OMVs (lane 3), and OMV sample (lane 4).
Mentions: To confirm the secretion of PrtV in association with OMVs, vesicles from strain C6706 were isolated and purified using an Optiprep density gradient centrifugation, as described in Materials and Methods. Analysis of the Optiprep fractions by immunoblotting using anti-PrtV polyclonal antibody revealed the presence of PrtV protein in fractions 7–10 (Fig 2A, upper panel). The presence of outer membrane protein in these fractions was confirmed by immunoblotting using anti-OmpU polyclonal antibody (Fig 2A, lower panel). As a control experiment, we analysed OMVs from the prtV mutant derivative of C6706 using the same approach. The absence of PrtV in the density gradient fractions was confirmed by immunoblotting, whereas OmpU was detected in fractions 5–10 (Fig 2B, upper and lower panels, respectively). Based on these observations we concluded that PrtV was secreted in association with vesicles. To estimate what percentage of the secreted PrtV was with associated with OMVs (i) total secreted PrtV in the cell-free culture supernatants (before OMV isolation); (ii) soluble PrtV (supernatant after separation of the OMVs by ultracentrifugation); and (iii) OMV-associated PrtV (purified vesicle sample) were examined for three independent cultures of the strain C6706 by immunoblotting. The immunoblot analysis of a representative set of samples is shown in Fig 2C. For each culture the amount of total secreted PrtV was given arbitrarily the value of 100. The results, given as a percentage, indicated that most of the secreted PrtV was associated with vesicles (70 ± 5%), whereas a smaller fraction was present in a free soluble form in the supernatant (30 ± 5%).

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