umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
The metalloprotease PrtV from Vibrio cholerae
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Show others and affiliations
2008 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 12, 3167-3177 p.Article in journal (Refereed) Published
Abstract [en]

The Vibrio metalloprotease PrtV was purified from the culture supernatant of a Vibrio cholerae derivative that is deficient in several other secreted peptidases, including the otherwise abundant hemagglutinin/protease HapA. The PrtV is synthesized as a 102 kDa protein, but undergoes several N- and C-terminal processing steps during V. cholerae envelope translocation and prolonged incubation. Purified V. cholerae PrtV protease forms of 81 or 73 kDa were stabilized by calcium ions. Removal of calcium resulted in further rapid autoproteolysis. The two major products of autoproteolysis of the PrtV protease were approximately 37 and 18 kDa and could not be separated under non-denaturing conditions, indicating they are interacting domains. In an assay using cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell death. Using human blood plasma as a source of potential substrates of mammalian origin for the PrtV protease, we found that the extracellular matrix components fibronectin and fibrinogen were degraded by the enzyme. Additional tests with individual protein substrates revealed that plasminogen was also a possible target for the PrtV protease.

Place, publisher, year, edition, pages
2008. Vol. 275, no 12, 3167-3177 p.
Keyword [en]
characterization, metalloprotease, PrtV, purification, V. cholerae
Identifiers
URN: urn:nbn:se:umu:diva-2862DOI: 10.1111/j.1742-4658.2008.06470.xOAI: oai:DiVA.org:umu-2862DiVA: diva2:141174
Note
i Karolis Vaitkevicius diss. utgör detta delarbete 2 med titel: Purification and characterization of the metalloprotease PrtV from Vibrio cholerae.Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Modulators of Vibrio cholerae predator interaction and virulence
Open this publication in new window or tab >>Modulators of Vibrio cholerae predator interaction and virulence
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor.  V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease.

Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane.  OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active.

V. cholerae strains are grouped into >200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease.  All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2009. 64 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1312
Keyword
Vibrio cholerae, Caenorhabditis elegans, PrtV, outer membrane vesicles, non-O1 non-O139, serum resistance
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-30211 (URN)978-91-7264-918-7 (ISBN)
Public defence
2010-01-22, Major Groove, Försörjningsvägen byggnad 6L, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2009-12-22 Created: 2009-12-14 Last updated: 2009-12-22Bibliographically approved
2. Effects of Vibrio cholerae protease and pigment production on environmental survival and host interaction
Open this publication in new window or tab >>Effects of Vibrio cholerae protease and pigment production on environmental survival and host interaction
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Only two out of more than 200 V. cholerae serogroups, classified on the basis of LPS structure, are associated with epidemic or pandemic cholera. These toxigenic serogroups carry phage-derived pathogenicity islands coding for the main virulence factors for establishment of cholera disease – cholera toxin (CTX) and toxin-coregulated pilus (TCP). The latter also serves as a bacterial surface receptor for the CTXΦ – the filamentous phage which carries the cholera toxin genes into otherwise harmless to human, environmental bacterium V. cholerae. In its natural aquatic habitat V. cholerae is subject to predator grazing, bacteriophage killing, temperature and pH changes, seasonality of plankton blooms and other environmental factors. Therefore understanding V. cholerae pathogenic and virulence potential requires the knowledge of its interaction not only with human host but also members of aquatic environment and environmental factors.

V. cholerae is capable of killing the nematode Caenorhabditis elegans. Using a reverse genetics approach, we demonstrated that the quorum sensing regulated protease PrtV is essential for this killing. Other proteases did not seem to contribute to virulence in this model. The data from this study suggest that the PrtV could be important to V. cholerae in its natural niche for its resistance to the grazing predators.

The PrtV protease belongs to an M6 family of metallopeptidases which is represented by an Immune Inhibitor A protease from the insect killing bacterium Bacillus thuringiensis. To characterize the protease in more detail, the PrtV was cloned, overexpressed in V. cholerae and purified from the culture supernatant. The enzyme was calcium stabilized and inhibited by metal ion chelators. In tests with in vitro cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell detachment and death. Using human blood plasma as a source of potential substrates, and by tests with purified candidate substrate proteins, we have identified fibrinogen (all α, β and γ chains), fibronectin and plasminogen to be degraded by the protease. Additionally, PrtV was found to alter the stability of V. cholerae cytolysin implicating its role in modulation of the reactogenicity of V. cholerae secreted factors.

Pigmentation has been considered to be important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Using transposon mutagenesis we identified the mutated locus of a pigment producing V. cholerae strain to encode a gene of a tyrosine catabolic pathway. The mutation in a putative homogentisate 1,2-dioxigenase gene lead to accumulation of homogentisic acid, its spontaneous oxidation and formation of a dark pigment. The pigment producing strain was altered in its ability to survive UV exposure and H2O2 stress, and was more efficient in colonizing the suckling mouse intestine compared to the wild type strain. Under the in vitro growth conditions the major virulence factor TcpA and CT expression was found to be somewhat enhanced too.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Medicinska fakulteten), 2007. 73 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1144
Keyword
Bacteria-host interaction, Nematode, Metalloprotease
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-1474 (URN)978-91-7264-464-9 (ISBN)
Public defence
2008-01-10, Major Groove, 6L, Norrlands Universitetssjukhus, Dept. Molecular Biology, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2010-01-26Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Vaitkevicius, KarolisRompikuntal, Pramod KumarLindmark, BarbroSong, TianyanWai, Sun Nyunt
By organisation
Molecular Biology (Faculty of Medicine)
In the same journal
The FEBS Journal

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 230 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf