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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

Immunoblot analysis of sub-cellular localization of PrtV protein in V. cholerae.(A) Immunoblot analyses of cell fractions from V. cholerae wild type strain C6706 (lanes 1, 3, 5, and 7) and the prtV mutant (lanes 2, 4, 6, and 8) using anti-PrtV serum (upper panel), anti-Crp antiserum (middle panel), and anti-β-lactamase antiserum (lower panel). Lanes 1 and 2: whole cell lysates; lanes 3 and 4, cytoplasmic fractions; lanes 5 and 6, periplasmic fractions; lanes 7 and 8, culture supernatants. Asterisks indicate the 81 kDa PrtV protein (B) Proteinase K susceptibility assay. OMVs from V. cholerae wild type strain C6706 were treated with 0.5 μg ml-1 of proteinase K (PK), 1% SDS and/or the proteinase K inhibitor PMSF (1 mM) as indicated. Samples were examined by immunoblot analysis using polyclonal anti-PrtV antiserum (upper panel). Lane 1: OMVs treated with only PK; lane 2: OMVs treated with SDS and PK; lane 3: OMVs treated with SDS, PMSF, and PK; lane 4: control OMVs. The same membrane was re-probed with OmpU antiserum as an internal control (lower panel).
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pone.0134098.g004: Immunoblot analysis of sub-cellular localization of PrtV protein in V. cholerae.(A) Immunoblot analyses of cell fractions from V. cholerae wild type strain C6706 (lanes 1, 3, 5, and 7) and the prtV mutant (lanes 2, 4, 6, and 8) using anti-PrtV serum (upper panel), anti-Crp antiserum (middle panel), and anti-β-lactamase antiserum (lower panel). Lanes 1 and 2: whole cell lysates; lanes 3 and 4, cytoplasmic fractions; lanes 5 and 6, periplasmic fractions; lanes 7 and 8, culture supernatants. Asterisks indicate the 81 kDa PrtV protein (B) Proteinase K susceptibility assay. OMVs from V. cholerae wild type strain C6706 were treated with 0.5 μg ml-1 of proteinase K (PK), 1% SDS and/or the proteinase K inhibitor PMSF (1 mM) as indicated. Samples were examined by immunoblot analysis using polyclonal anti-PrtV antiserum (upper panel). Lane 1: OMVs treated with only PK; lane 2: OMVs treated with SDS and PK; lane 3: OMVs treated with SDS, PMSF, and PK; lane 4: control OMVs. The same membrane was re-probed with OmpU antiserum as an internal control (lower panel).

Mentions: To analyze how PrtV may be released from the bacterial cell via OMVs, we first determined the subcellular localization of the PrtV protein in the C6706/pBAD18 and C6706/pBAD::prtV strains using a fractionation assay. According to our findings, full-length, 81 kDa PrtV was present in the whole cell (Fig 4A, lane 1), cytoplasmic (Fig 4A, lane 3), and extracellular fractions (Fig 4A, lane 7), but not in the periplasmic fraction (Fig 4A, lane 5). Crp and β-lactamase were used as marker proteins to confirm the cytoplasmic and periplasmic content, respectively, of the fractions. The 37 kDa form of PrtV was abundant in the periplasmic fraction (Fig 4A, lane 5) and in the OMV fraction (Fig 3B, upper panel, lane 1), suggesting that the full-length protein is subject to proteolytic cleavage in the periplasmic space. Moreover, it could be hypothesized that the 37 kDa form is packaged as a part of the OMV luminal content during vesicle biogenesis. Our hypothesis was also supported by the Fig 2C results in which the 37kDa form is only in the OMV, not found in the supernatant.


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)

Immunoblot analysis of sub-cellular localization of PrtV protein in V. cholerae.(A) Immunoblot analyses of cell fractions from V. cholerae wild type strain C6706 (lanes 1, 3, 5, and 7) and the prtV mutant (lanes 2, 4, 6, and 8) using anti-PrtV serum (upper panel), anti-Crp antiserum (middle panel), and anti-β-lactamase antiserum (lower panel). Lanes 1 and 2: whole cell lysates; lanes 3 and 4, cytoplasmic fractions; lanes 5 and 6, periplasmic fractions; lanes 7 and 8, culture supernatants. Asterisks indicate the 81 kDa PrtV protein (B) Proteinase K susceptibility assay. OMVs from V. cholerae wild type strain C6706 were treated with 0.5 μg ml-1 of proteinase K (PK), 1% SDS and/or the proteinase K inhibitor PMSF (1 mM) as indicated. Samples were examined by immunoblot analysis using polyclonal anti-PrtV antiserum (upper panel). Lane 1: OMVs treated with only PK; lane 2: OMVs treated with SDS and PK; lane 3: OMVs treated with SDS, PMSF, and PK; lane 4: control OMVs. The same membrane was re-probed with OmpU antiserum as an internal control (lower panel).
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Related In: Results  -  Collection

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pone.0134098.g004: Immunoblot analysis of sub-cellular localization of PrtV protein in V. cholerae.(A) Immunoblot analyses of cell fractions from V. cholerae wild type strain C6706 (lanes 1, 3, 5, and 7) and the prtV mutant (lanes 2, 4, 6, and 8) using anti-PrtV serum (upper panel), anti-Crp antiserum (middle panel), and anti-β-lactamase antiserum (lower panel). Lanes 1 and 2: whole cell lysates; lanes 3 and 4, cytoplasmic fractions; lanes 5 and 6, periplasmic fractions; lanes 7 and 8, culture supernatants. Asterisks indicate the 81 kDa PrtV protein (B) Proteinase K susceptibility assay. OMVs from V. cholerae wild type strain C6706 were treated with 0.5 μg ml-1 of proteinase K (PK), 1% SDS and/or the proteinase K inhibitor PMSF (1 mM) as indicated. Samples were examined by immunoblot analysis using polyclonal anti-PrtV antiserum (upper panel). Lane 1: OMVs treated with only PK; lane 2: OMVs treated with SDS and PK; lane 3: OMVs treated with SDS, PMSF, and PK; lane 4: control OMVs. The same membrane was re-probed with OmpU antiserum as an internal control (lower panel).
Mentions: To analyze how PrtV may be released from the bacterial cell via OMVs, we first determined the subcellular localization of the PrtV protein in the C6706/pBAD18 and C6706/pBAD::prtV strains using a fractionation assay. According to our findings, full-length, 81 kDa PrtV was present in the whole cell (Fig 4A, lane 1), cytoplasmic (Fig 4A, lane 3), and extracellular fractions (Fig 4A, lane 7), but not in the periplasmic fraction (Fig 4A, lane 5). Crp and β-lactamase were used as marker proteins to confirm the cytoplasmic and periplasmic content, respectively, of the fractions. The 37 kDa form of PrtV was abundant in the periplasmic fraction (Fig 4A, lane 5) and in the OMV fraction (Fig 3B, upper panel, lane 1), suggesting that the full-length protein is subject to proteolytic cleavage in the periplasmic space. Moreover, it could be hypothesized that the 37 kDa form is packaged as a part of the OMV luminal content during vesicle biogenesis. Our hypothesis was also supported by the Fig 2C results in which the 37kDa form is only in the OMV, not found in the supernatant.

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