Limits...
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

V. cholerae OMVs internalization into HCT8 cells.OMVs from the wild type strain C6706 and the prtV mutant were labeled with PKH26 red fluorescence marker and subsequently the HCT8 cells were treated for 6 hrs with buffer (A), PKH26-labeled OMVs from V. cholerae wild type strain C6706 (B) or with OMVs from the prtV mutant (C). After the treatment, cells were fixed and actin filaments and nuclei were stained with phalloidin and DAPI, respectively. Internalized OMVs are indicated with white arrows. Confocal Z-stack projections are shown in all images. The crosshairs indicate the positions of the xz and yz planes. Bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4519245&req=5

pone.0134098.g008: V. cholerae OMVs internalization into HCT8 cells.OMVs from the wild type strain C6706 and the prtV mutant were labeled with PKH26 red fluorescence marker and subsequently the HCT8 cells were treated for 6 hrs with buffer (A), PKH26-labeled OMVs from V. cholerae wild type strain C6706 (B) or with OMVs from the prtV mutant (C). After the treatment, cells were fixed and actin filaments and nuclei were stained with phalloidin and DAPI, respectively. Internalized OMVs are indicated with white arrows. Confocal Z-stack projections are shown in all images. The crosshairs indicate the positions of the xz and yz planes. Bars represent 10 μm.

Mentions: To investigate if OMVs isolated from V. cholerae can internalize into HCT8 cells, confocal microscopy analyses were performed for the detection of the internalized vesicles. For this purpose, we labeled samples of OMVs isolated from the wild type V. cholerae strain C6706 and the prtV mutant containing 3.5 x 1011 and 4 x 1011 OMV-particles, respectively, with a red fluorochrome, PKH26. The use of this fluorescent marker to monitor cell trafficking and function has been well documented in earlier studies [37–40]. After incubating the cells with OMV samples (either WT C6706 or the prtV mutant) for 1 h, we observed that several wild type OMVs, appearing as red dots surrounding the nuclei of the effected cells (Fig 8B). Similarly, OMVs obtained from the prtV mutant were internalized into the HCT8 cells (Fig 8C). Based on these observations we concluded that there was indeed internalization V. cholerae OMVs were internalized into the HCT8 cells regardless of the presence of PrtV.


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)

V. cholerae OMVs internalization into HCT8 cells.OMVs from the wild type strain C6706 and the prtV mutant were labeled with PKH26 red fluorescence marker and subsequently the HCT8 cells were treated for 6 hrs with buffer (A), PKH26-labeled OMVs from V. cholerae wild type strain C6706 (B) or with OMVs from the prtV mutant (C). After the treatment, cells were fixed and actin filaments and nuclei were stained with phalloidin and DAPI, respectively. Internalized OMVs are indicated with white arrows. Confocal Z-stack projections are shown in all images. The crosshairs indicate the positions of the xz and yz planes. Bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134098.g008: V. cholerae OMVs internalization into HCT8 cells.OMVs from the wild type strain C6706 and the prtV mutant were labeled with PKH26 red fluorescence marker and subsequently the HCT8 cells were treated for 6 hrs with buffer (A), PKH26-labeled OMVs from V. cholerae wild type strain C6706 (B) or with OMVs from the prtV mutant (C). After the treatment, cells were fixed and actin filaments and nuclei were stained with phalloidin and DAPI, respectively. Internalized OMVs are indicated with white arrows. Confocal Z-stack projections are shown in all images. The crosshairs indicate the positions of the xz and yz planes. Bars represent 10 μm.
Mentions: To investigate if OMVs isolated from V. cholerae can internalize into HCT8 cells, confocal microscopy analyses were performed for the detection of the internalized vesicles. For this purpose, we labeled samples of OMVs isolated from the wild type V. cholerae strain C6706 and the prtV mutant containing 3.5 x 1011 and 4 x 1011 OMV-particles, respectively, with a red fluorochrome, PKH26. The use of this fluorescent marker to monitor cell trafficking and function has been well documented in earlier studies [37–40]. After incubating the cells with OMV samples (either WT C6706 or the prtV mutant) for 1 h, we observed that several wild type OMVs, appearing as red dots surrounding the nuclei of the effected cells (Fig 8B). Similarly, OMVs obtained from the prtV mutant were internalized into the HCT8 cells (Fig 8C). Based on these observations we concluded that there was indeed internalization V. cholerae OMVs were internalized into the HCT8 cells regardless of the presence of PrtV.

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