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Effects of Vibrio cholerae protease and pigment production on environmental survival and host interaction
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
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 [en]
Bacteria-host interaction, Nematode, Metalloprotease
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-1474ISBN: 978-91-7264-464-9 (print)OAI: oai:DiVA.org:umu-1474DiVA: diva2:141177
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
List of papers
1. A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing
Open this publication in new window or tab >>A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 24, 9280-9285 p.Article in journal (Refereed) Published
Abstract [en]

Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.

Keyword
Animals, Bacterial Proteins/genetics/metabolism, Biofilms, Caenorhabditis elegans/cytology/microbiology/physiology, Cell Communication, Cell Line; Tumor, Cholera Toxin/metabolism, Feeding Behavior, Fimbriae; Bacterial/metabolism, Humans, Interleukin-8/secretion, Intestines/cytology/microbiology, Peptide Hydrolases/genetics/metabolism, Predatory Behavior, Repressor Proteins/genetics/metabolism, Survival Rate, Trans-Activators/genetics/metabolism, Transcription Factors/genetics/metabolism, Vibrio cholerae/enzymology/genetics/pathogenicity
National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-6243 (URN)10.1073/pnas.0601754103 (DOI)16754867 (PubMedID)
Available from: 2008-12-14 Created: 2008-12-14 Last updated: 2017-10-24Bibliographically approved
2. The metalloprotease PrtV from Vibrio cholerae
Open this publication in new window or tab >>The metalloprotease PrtV from Vibrio cholerae
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2008 (English)In: The FEBS Journal, ISSN 1742-464X, 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.

Keyword
characterization, metalloprotease, PrtV, purification, V. cholerae
Identifiers
urn:nbn:se:umu:diva-2862 (URN)10.1111/j.1742-4658.2008.06470.x (DOI)
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: 2015-03-05Bibliographically approved
3. The PrtV protease in Vibrio cholerae influenses expression of the VCC/HlyA cytolytic activity
Open this publication in new window or tab >>The PrtV protease in Vibrio cholerae influenses expression of the VCC/HlyA cytolytic activity
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-30981 (URN)
Note
Manuscript in preparation.Available from: 2010-01-26 Created: 2010-01-26 Last updated: 2015-03-05
4. Genetic analysis of a pigment producing mutant of Vibrio cholerae
Open this publication in new window or tab >>Genetic analysis of a pigment producing mutant of Vibrio cholerae
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2864 (URN)
Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2010-01-13Bibliographically approved

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