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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

PrtV secretion in type I secretion mutants of V. cholerae O1 El Tor strain C6706.(A) PrtV secretion was analyzed using culture supernatants of V. cholerae wild type strain C6706 and its mutant derivatives. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (B) A Coomassie blue stained gel as a control for sample loading. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (C) PrtV secretion in type II secretion system mutants of V. cholerae O1 El Tor strains. Lanes 1 and 2: culture supernatants of wild type C6706 and ΔprtV; lanes 3, 4 and 5: culture supernatants of wild type 3083, ΔepsC and ΔepsC strain carrying the cloned epsC allele; lanes 6, 7 and 8: culture supernatants of wild type TRH7000, ΔepsC and ΔepsC strain carrying the cloned epsC allele.
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pone.0134098.g006: PrtV secretion in type I secretion mutants of V. cholerae O1 El Tor strain C6706.(A) PrtV secretion was analyzed using culture supernatants of V. cholerae wild type strain C6706 and its mutant derivatives. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (B) A Coomassie blue stained gel as a control for sample loading. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (C) PrtV secretion in type II secretion system mutants of V. cholerae O1 El Tor strains. Lanes 1 and 2: culture supernatants of wild type C6706 and ΔprtV; lanes 3, 4 and 5: culture supernatants of wild type 3083, ΔepsC and ΔepsC strain carrying the cloned epsC allele; lanes 6, 7 and 8: culture supernatants of wild type TRH7000, ΔepsC and ΔepsC strain carrying the cloned epsC allele.

Mentions: Our findings prompted us to determine which secretion system might be involved in secretion of PrtV through the outer membrane and thereafter into the culture supernatant and/or OMVs. To test if the type I secretion system is needed for PrtV secretion, we constructed tolC and hlyD in-frame deletion mutants of V. cholerae wild type strain C6706 because the TolC and HlyD proteins are essential components of the type I secretion system of bacteria [35]. We compared secretion of PrtV in the tolC and the hlyD mutants with the wild type strain C6706 by immunoblot analysis. We observed no difference in the levels of secreted PrtV in the wild type and the mutants (Fig 6A, lanes 1–3). A Coomassie blue stained gel was included to verify equal sample loading (Fig 6B).


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)

PrtV secretion in type I secretion mutants of V. cholerae O1 El Tor strain C6706.(A) PrtV secretion was analyzed using culture supernatants of V. cholerae wild type strain C6706 and its mutant derivatives. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (B) A Coomassie blue stained gel as a control for sample loading. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (C) PrtV secretion in type II secretion system mutants of V. cholerae O1 El Tor strains. Lanes 1 and 2: culture supernatants of wild type C6706 and ΔprtV; lanes 3, 4 and 5: culture supernatants of wild type 3083, ΔepsC and ΔepsC strain carrying the cloned epsC allele; lanes 6, 7 and 8: culture supernatants of wild type TRH7000, ΔepsC and ΔepsC strain carrying the cloned epsC allele.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519245&req=5

pone.0134098.g006: PrtV secretion in type I secretion mutants of V. cholerae O1 El Tor strain C6706.(A) PrtV secretion was analyzed using culture supernatants of V. cholerae wild type strain C6706 and its mutant derivatives. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (B) A Coomassie blue stained gel as a control for sample loading. Lane 1: C6706; lane 2: ΔtolC; lane 3: ΔhlyD; lane 4: ΔprtV. (C) PrtV secretion in type II secretion system mutants of V. cholerae O1 El Tor strains. Lanes 1 and 2: culture supernatants of wild type C6706 and ΔprtV; lanes 3, 4 and 5: culture supernatants of wild type 3083, ΔepsC and ΔepsC strain carrying the cloned epsC allele; lanes 6, 7 and 8: culture supernatants of wild type TRH7000, ΔepsC and ΔepsC strain carrying the cloned epsC allele.
Mentions: Our findings prompted us to determine which secretion system might be involved in secretion of PrtV through the outer membrane and thereafter into the culture supernatant and/or OMVs. To test if the type I secretion system is needed for PrtV secretion, we constructed tolC and hlyD in-frame deletion mutants of V. cholerae wild type strain C6706 because the TolC and HlyD proteins are essential components of the type I secretion system of bacteria [35]. We compared secretion of PrtV in the tolC and the hlyD mutants with the wild type strain C6706 by immunoblot analysis. We observed no difference in the levels of secreted PrtV in the wild type and the mutants (Fig 6A, lanes 1–3). A Coomassie blue stained gel was included to verify equal sample loading (Fig 6B).

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