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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

Analyses of biological activities of PrtV.HCT8 cells were treated with 50 μl OMVs (total protein concentration 60 μg/ml) from the wild type V. cholerae strain C6706 and the prtV mutant. Cells were treated with 20 mM Tris-HCl as a negative control (A, E), with OMVs from the wild type strain C6706 (B and F) or from the prtV mutant (C and G) or from the the prtV mutant/pΔPKD PrtV (D and H) or with 20 nM purified PrtV protein for 6 h as a positive control (I). The treatment was performed for 6 h (A, B, C, D) and for 12 h (E, F, G, H). Bars represent 10 μm.
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pone.0134098.g007: Analyses of biological activities of PrtV.HCT8 cells were treated with 50 μl OMVs (total protein concentration 60 μg/ml) from the wild type V. cholerae strain C6706 and the prtV mutant. Cells were treated with 20 mM Tris-HCl as a negative control (A, E), with OMVs from the wild type strain C6706 (B and F) or from the prtV mutant (C and G) or from the the prtV mutant/pΔPKD PrtV (D and H) or with 20 nM purified PrtV protein for 6 h as a positive control (I). The treatment was performed for 6 h (A, B, C, D) and for 12 h (E, F, G, H). Bars represent 10 μm.

Mentions: In our earlier studies, we demonstrated that PrtV is an active protease as it is able to cleave proteins such as fibrinogen, fibronectin and plasminogen. We also showed that purified PrtV exhibited a dose-dependent cytotoxic activity towards mammalian cells [9]. To test the biological activity of vesicle-associated PrtV, we incubated HCT8 cells with OMVs obtained from the wild type strain C6706, the prtV mutant, and the strain expressed PrtVΔPKD. According to our findings, no apparent morphological changes of the epithelial cells were observed when the cells were treated with OMVs from these strains for 6 h (Fig 7B, 7C and 7D). However, after 12 h incubation, rounding and detachment of the HCT8 cells was observed when incubated with OMVs from both the wild type strain and the strain expressed PrtVΔPKD (Fig 7F and 7H). A similar result was obtained when the cells were treated with purified PrtV (20 nM) for 6 h (Fig 7I). In contrast, such morphological effects on the cells were not observed when the cells were treated with vesicles isolated from the ΔprtV mutant (Fig 7C and 7G) or with buffer (Fig 7A and 7E). Thus, based on these results we concluded that OMV-associated full length PrtV and PrtVΔPKD1-2 were both biologically active. Moreover, taking into consideration that OMVs isolated from the bacterial strain harboring only PrtVΔPKD alle contain only 37 kDa form suggesting that the biological activity that we observed might be mainly due to the action of 37 kDa form.


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)

Analyses of biological activities of PrtV.HCT8 cells were treated with 50 μl OMVs (total protein concentration 60 μg/ml) from the wild type V. cholerae strain C6706 and the prtV mutant. Cells were treated with 20 mM Tris-HCl as a negative control (A, E), with OMVs from the wild type strain C6706 (B and F) or from the prtV mutant (C and G) or from the the prtV mutant/pΔPKD PrtV (D and H) or with 20 nM purified PrtV protein for 6 h as a positive control (I). The treatment was performed for 6 h (A, B, C, D) and for 12 h (E, F, G, H). Bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134098.g007: Analyses of biological activities of PrtV.HCT8 cells were treated with 50 μl OMVs (total protein concentration 60 μg/ml) from the wild type V. cholerae strain C6706 and the prtV mutant. Cells were treated with 20 mM Tris-HCl as a negative control (A, E), with OMVs from the wild type strain C6706 (B and F) or from the prtV mutant (C and G) or from the the prtV mutant/pΔPKD PrtV (D and H) or with 20 nM purified PrtV protein for 6 h as a positive control (I). The treatment was performed for 6 h (A, B, C, D) and for 12 h (E, F, G, H). Bars represent 10 μm.
Mentions: In our earlier studies, we demonstrated that PrtV is an active protease as it is able to cleave proteins such as fibrinogen, fibronectin and plasminogen. We also showed that purified PrtV exhibited a dose-dependent cytotoxic activity towards mammalian cells [9]. To test the biological activity of vesicle-associated PrtV, we incubated HCT8 cells with OMVs obtained from the wild type strain C6706, the prtV mutant, and the strain expressed PrtVΔPKD. According to our findings, no apparent morphological changes of the epithelial cells were observed when the cells were treated with OMVs from these strains for 6 h (Fig 7B, 7C and 7D). However, after 12 h incubation, rounding and detachment of the HCT8 cells was observed when incubated with OMVs from both the wild type strain and the strain expressed PrtVΔPKD (Fig 7F and 7H). A similar result was obtained when the cells were treated with purified PrtV (20 nM) for 6 h (Fig 7I). In contrast, such morphological effects on the cells were not observed when the cells were treated with vesicles isolated from the ΔprtV mutant (Fig 7C and 7G) or with buffer (Fig 7A and 7E). Thus, based on these results we concluded that OMV-associated full length PrtV and PrtVΔPKD1-2 were both biologically active. Moreover, taking into consideration that OMVs isolated from the bacterial strain harboring only PrtVΔPKD alle contain only 37 kDa form suggesting that the biological activity that we observed might be mainly due to the action of 37 kDa form.

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