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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 PrtV expression and secretion and ultrastructural analysis of V. cholerae surface structures.(A) Immunoblot analysis of expression and secretion of PrtV in different V. cholerae O1 isolates. Bacterial strains were grown at 30°C and samples were collected at OD600 2.0. Samples of whole cell extracts from overnight cultures (lanes 1–3, 5 μl) and culture supernatants (lanes 4–6, 10 μl, corresponding to tenfold concentration compared with the whole cell samples) were loaded in the gel. Immunoblotting was done using anti-PrtV polyclonal antiserum. Lanes 1–3; whole cell lysates; lanes 4–6: culture supernatants from wild type V. cholerae El Tor O1 strains A1552, C6706, and P27459 respectively. (B) Ultrastructural analysis of V. cholerae by electron microscopy. An electron micrograph showing the flagella (open arrows) and OMVs (closed arrows). Bar, 500 nm. (C) PrtV association with OMVs from different V. cholerae isolates. Immunoblot analysis of OMVs from different V. cholerae isolates using PrtV polyclonal antiserum. Bacterial strains were grown at 30°C for 16 h and OMVs were isolated using the procedure described in Materials and Methods. 10 μl of OMV samples were loaded for immunoblot analyses and SDS-PAGE analyses by Coomassie blue staining. Lanes 1–6; OMVs from V. cholerae non-O1/non-O139 serogroup: V:52, V:5/04, V:6/04, KI17036, 93Ag19, and NAGV6; lanes 7–9: OMVs from V. cholerae O1 El Tor clinical isolates: P27459, C6706, and A1552; lane 10: OMVs from V. cholerae classical O1 strain 569B; lanes 11–13: OMVs from V. cholerae O1 environmental isolates: AJ4, AJ3, and AJ2. Lane 14, C6706 ΔprtV mutant.
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pone.0134098.g001: Immunoblot analyses of PrtV expression and secretion and ultrastructural analysis of V. cholerae surface structures.(A) Immunoblot analysis of expression and secretion of PrtV in different V. cholerae O1 isolates. Bacterial strains were grown at 30°C and samples were collected at OD600 2.0. Samples of whole cell extracts from overnight cultures (lanes 1–3, 5 μl) and culture supernatants (lanes 4–6, 10 μl, corresponding to tenfold concentration compared with the whole cell samples) were loaded in the gel. Immunoblotting was done using anti-PrtV polyclonal antiserum. Lanes 1–3; whole cell lysates; lanes 4–6: culture supernatants from wild type V. cholerae El Tor O1 strains A1552, C6706, and P27459 respectively. (B) Ultrastructural analysis of V. cholerae by electron microscopy. An electron micrograph showing the flagella (open arrows) and OMVs (closed arrows). Bar, 500 nm. (C) PrtV association with OMVs from different V. cholerae isolates. Immunoblot analysis of OMVs from different V. cholerae isolates using PrtV polyclonal antiserum. Bacterial strains were grown at 30°C for 16 h and OMVs were isolated using the procedure described in Materials and Methods. 10 μl of OMV samples were loaded for immunoblot analyses and SDS-PAGE analyses by Coomassie blue staining. Lanes 1–6; OMVs from V. cholerae non-O1/non-O139 serogroup: V:52, V:5/04, V:6/04, KI17036, 93Ag19, and NAGV6; lanes 7–9: OMVs from V. cholerae O1 El Tor clinical isolates: P27459, C6706, and A1552; lane 10: OMVs from V. cholerae classical O1 strain 569B; lanes 11–13: OMVs from V. cholerae O1 environmental isolates: AJ4, AJ3, and AJ2. Lane 14, C6706 ΔprtV mutant.

Mentions: In our earlier studies, we have shown that PrtV was secreted into culture supernatants of the V. cholerae wild type strain C6706 as a 102 kDa protein that due to autoproteolytic cleavages also resulted in two shorter forms (81 kDa and 37 kDa, respectively) with protease activity [9]. It was suggested that all three forms are physiologically important. Immunoblot analysis was used to confirm that PrtV is secreted by additional V. cholerae strains, i.e. the O1 El Tor strains A1552 and P27459 (Fig 1A). Electron microscopy analysis of strain C6706 revealed the presence of OMVs surrounding the bacterial cells (Fig 1B). As cell supernatants samples include not only soluble extracellular proteins, but also OMVs, we sought to assess if PrtV may be associated with OMVs in V. cholerae. Vesicles were isolated from overnight cultures (16 h) of a selection of strains as described in Material and Methods. The two forms of PrtV protein (81 kDa and 37 kDa) were detected by immunoblot in association with OMVs obtained from ten out of thirteen tested strains, i.e. from V. cholerae non-O1/non-O139 strainsV:5/04, V:6/04, KI17036, 93Ag19 and NAGV6 (Fig 1C, lanes 2–6); from O1 El Tor clinical isolates C6706 and A1552 (Fig 1C, lanes 8–9); from classical O1 strain 569B (Fig 1C, lane 10) and from O1 environmental isolates AJ4, AJ3 and AJ2 (Fig 1C, lanes 11–13). Interestingly, the non-O1/non-O139 V. cholerae strain V52 (Fig 1C, lane 1) and the O1 El Tor strain P27459 (Fig 1C, lane 7) have only one form of PrtV protein, the 81 kDa and 37 kDa form respectively. A Coomassie blue stained gel was shown to estimate the loading amount of each sample (Fig 1D) Based on these observations we propose that secretion of PrtV via OMVs may be common among V. cholerae strains.


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 PrtV expression and secretion and ultrastructural analysis of V. cholerae surface structures.(A) Immunoblot analysis of expression and secretion of PrtV in different V. cholerae O1 isolates. Bacterial strains were grown at 30°C and samples were collected at OD600 2.0. Samples of whole cell extracts from overnight cultures (lanes 1–3, 5 μl) and culture supernatants (lanes 4–6, 10 μl, corresponding to tenfold concentration compared with the whole cell samples) were loaded in the gel. Immunoblotting was done using anti-PrtV polyclonal antiserum. Lanes 1–3; whole cell lysates; lanes 4–6: culture supernatants from wild type V. cholerae El Tor O1 strains A1552, C6706, and P27459 respectively. (B) Ultrastructural analysis of V. cholerae by electron microscopy. An electron micrograph showing the flagella (open arrows) and OMVs (closed arrows). Bar, 500 nm. (C) PrtV association with OMVs from different V. cholerae isolates. Immunoblot analysis of OMVs from different V. cholerae isolates using PrtV polyclonal antiserum. Bacterial strains were grown at 30°C for 16 h and OMVs were isolated using the procedure described in Materials and Methods. 10 μl of OMV samples were loaded for immunoblot analyses and SDS-PAGE analyses by Coomassie blue staining. Lanes 1–6; OMVs from V. cholerae non-O1/non-O139 serogroup: V:52, V:5/04, V:6/04, KI17036, 93Ag19, and NAGV6; lanes 7–9: OMVs from V. cholerae O1 El Tor clinical isolates: P27459, C6706, and A1552; lane 10: OMVs from V. cholerae classical O1 strain 569B; lanes 11–13: OMVs from V. cholerae O1 environmental isolates: AJ4, AJ3, and AJ2. Lane 14, C6706 ΔprtV mutant.
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Related In: Results  -  Collection

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pone.0134098.g001: Immunoblot analyses of PrtV expression and secretion and ultrastructural analysis of V. cholerae surface structures.(A) Immunoblot analysis of expression and secretion of PrtV in different V. cholerae O1 isolates. Bacterial strains were grown at 30°C and samples were collected at OD600 2.0. Samples of whole cell extracts from overnight cultures (lanes 1–3, 5 μl) and culture supernatants (lanes 4–6, 10 μl, corresponding to tenfold concentration compared with the whole cell samples) were loaded in the gel. Immunoblotting was done using anti-PrtV polyclonal antiserum. Lanes 1–3; whole cell lysates; lanes 4–6: culture supernatants from wild type V. cholerae El Tor O1 strains A1552, C6706, and P27459 respectively. (B) Ultrastructural analysis of V. cholerae by electron microscopy. An electron micrograph showing the flagella (open arrows) and OMVs (closed arrows). Bar, 500 nm. (C) PrtV association with OMVs from different V. cholerae isolates. Immunoblot analysis of OMVs from different V. cholerae isolates using PrtV polyclonal antiserum. Bacterial strains were grown at 30°C for 16 h and OMVs were isolated using the procedure described in Materials and Methods. 10 μl of OMV samples were loaded for immunoblot analyses and SDS-PAGE analyses by Coomassie blue staining. Lanes 1–6; OMVs from V. cholerae non-O1/non-O139 serogroup: V:52, V:5/04, V:6/04, KI17036, 93Ag19, and NAGV6; lanes 7–9: OMVs from V. cholerae O1 El Tor clinical isolates: P27459, C6706, and A1552; lane 10: OMVs from V. cholerae classical O1 strain 569B; lanes 11–13: OMVs from V. cholerae O1 environmental isolates: AJ4, AJ3, and AJ2. Lane 14, C6706 ΔprtV mutant.
Mentions: In our earlier studies, we have shown that PrtV was secreted into culture supernatants of the V. cholerae wild type strain C6706 as a 102 kDa protein that due to autoproteolytic cleavages also resulted in two shorter forms (81 kDa and 37 kDa, respectively) with protease activity [9]. It was suggested that all three forms are physiologically important. Immunoblot analysis was used to confirm that PrtV is secreted by additional V. cholerae strains, i.e. the O1 El Tor strains A1552 and P27459 (Fig 1A). Electron microscopy analysis of strain C6706 revealed the presence of OMVs surrounding the bacterial cells (Fig 1B). As cell supernatants samples include not only soluble extracellular proteins, but also OMVs, we sought to assess if PrtV may be associated with OMVs in V. cholerae. Vesicles were isolated from overnight cultures (16 h) of a selection of strains as described in Material and Methods. The two forms of PrtV protein (81 kDa and 37 kDa) were detected by immunoblot in association with OMVs obtained from ten out of thirteen tested strains, i.e. from V. cholerae non-O1/non-O139 strainsV:5/04, V:6/04, KI17036, 93Ag19 and NAGV6 (Fig 1C, lanes 2–6); from O1 El Tor clinical isolates C6706 and A1552 (Fig 1C, lanes 8–9); from classical O1 strain 569B (Fig 1C, lane 10) and from O1 environmental isolates AJ4, AJ3 and AJ2 (Fig 1C, lanes 11–13). Interestingly, the non-O1/non-O139 V. cholerae strain V52 (Fig 1C, lane 1) and the O1 El Tor strain P27459 (Fig 1C, lane 7) have only one form of PrtV protein, the 81 kDa and 37 kDa form respectively. A Coomassie blue stained gel was shown to estimate the loading amount of each sample (Fig 1D) Based on these observations we propose that secretion of PrtV via OMVs may be common among V. cholerae strains.

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