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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 analyses of the wild type PrtV protein and its PKD domain deletion mutant in culture supernatants and OMV samples.Immunoblot analysis was performed using anti-PrtV antiserum (A) and anti-OmpU antiserum (B) with the following samples: lanes 1–3: V. cholerae ΔprtV strain carrying the cloned wild type allele of prtV; lanes 4–6: the ΔprtV strain carrying the cloned prtVΔPKD allele; lanes 7–9: the ΔprtV strain carrying the pBAD18 cloning vector. Lanes 1, 4 and 7 were loaded with supernatant samples before ultracentrifugation (Sup1). Lanes 2, 5 and 8 were loaded with the supernatants after ultracentrifugation (Sup2). Lanes 3, 6 and 9 were loaded with the OMV samples.
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pone.0134098.g005: Immunoblot analyses of the wild type PrtV protein and its PKD domain deletion mutant in culture supernatants and OMV samples.Immunoblot analysis was performed using anti-PrtV antiserum (A) and anti-OmpU antiserum (B) with the following samples: lanes 1–3: V. cholerae ΔprtV strain carrying the cloned wild type allele of prtV; lanes 4–6: the ΔprtV strain carrying the cloned prtVΔPKD allele; lanes 7–9: the ΔprtV strain carrying the pBAD18 cloning vector. Lanes 1, 4 and 7 were loaded with supernatant samples before ultracentrifugation (Sup1). Lanes 2, 5 and 8 were loaded with the supernatants after ultracentrifugation (Sup2). Lanes 3, 6 and 9 were loaded with the OMV samples.

Mentions: In order to test the role of the PKD-domains in OMV-associated secretion of PrtV, we constructed expression plasmids encoding either the wild type prtV gene or a prtVΔPKD1-2 allele. These clones and the empty pBAD18 vector were introduced into the prtV mutant of V. cholerae strain C6706. Bacterial culture supernatants before and after ultracentrifugation, and OMVs were isolated from these three strains, and the OMV-associated secretion of PrtV and PrtVΔPKD1-2 was analyzed by immunoblotting. Unlike full-length PrtV, the full-length PrtVΔPKD1-2 protein was not detected in association with OMVs (Fig 5A, lane 6) although the full-length PrtVΔPKD1-2 was observed in the supernatants, indicating that it was stable and translocated from the bacterial cells grown in LB media containing 50 μg/ml carbenicillin and 0.01% arabinose. Interestingly, the processed 37 kDa form of PrtV could be detected in OMVs regardless if the strain expressed wild-type PrtV or PrtVΔPKD1-2 (Fig 5A, lanes 3 and 6). As a control, OmpU immunoblot detection was shown (Fig 5B). Taken together, the findings suggested that the PKD domains might have a role for secreted full-length PrtV in its association with OMVs.


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 analyses of the wild type PrtV protein and its PKD domain deletion mutant in culture supernatants and OMV samples.Immunoblot analysis was performed using anti-PrtV antiserum (A) and anti-OmpU antiserum (B) with the following samples: lanes 1–3: V. cholerae ΔprtV strain carrying the cloned wild type allele of prtV; lanes 4–6: the ΔprtV strain carrying the cloned prtVΔPKD allele; lanes 7–9: the ΔprtV strain carrying the pBAD18 cloning vector. Lanes 1, 4 and 7 were loaded with supernatant samples before ultracentrifugation (Sup1). Lanes 2, 5 and 8 were loaded with the supernatants after ultracentrifugation (Sup2). Lanes 3, 6 and 9 were loaded with the OMV samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519245&req=5

pone.0134098.g005: Immunoblot analyses of the wild type PrtV protein and its PKD domain deletion mutant in culture supernatants and OMV samples.Immunoblot analysis was performed using anti-PrtV antiserum (A) and anti-OmpU antiserum (B) with the following samples: lanes 1–3: V. cholerae ΔprtV strain carrying the cloned wild type allele of prtV; lanes 4–6: the ΔprtV strain carrying the cloned prtVΔPKD allele; lanes 7–9: the ΔprtV strain carrying the pBAD18 cloning vector. Lanes 1, 4 and 7 were loaded with supernatant samples before ultracentrifugation (Sup1). Lanes 2, 5 and 8 were loaded with the supernatants after ultracentrifugation (Sup2). Lanes 3, 6 and 9 were loaded with the OMV samples.
Mentions: In order to test the role of the PKD-domains in OMV-associated secretion of PrtV, we constructed expression plasmids encoding either the wild type prtV gene or a prtVΔPKD1-2 allele. These clones and the empty pBAD18 vector were introduced into the prtV mutant of V. cholerae strain C6706. Bacterial culture supernatants before and after ultracentrifugation, and OMVs were isolated from these three strains, and the OMV-associated secretion of PrtV and PrtVΔPKD1-2 was analyzed by immunoblotting. Unlike full-length PrtV, the full-length PrtVΔPKD1-2 protein was not detected in association with OMVs (Fig 5A, lane 6) although the full-length PrtVΔPKD1-2 was observed in the supernatants, indicating that it was stable and translocated from the bacterial cells grown in LB media containing 50 μg/ml carbenicillin and 0.01% arabinose. Interestingly, the processed 37 kDa form of PrtV could be detected in OMVs regardless if the strain expressed wild-type PrtV or PrtVΔPKD1-2 (Fig 5A, lanes 3 and 6). As a control, OmpU immunoblot detection was shown (Fig 5B). Taken together, the findings suggested that the PKD domains might have a role for secreted full-length PrtV in its association with OMVs.

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