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Modulators of Vibrio cholerae predator interaction and virulence
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Sun Nyunt Wai)
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 [en]
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: urn:nbn:se:umu:diva-30211ISBN: 978-91-7264-918-7 (print)OAI: oai:DiVA.org:umu-30211DiVA: diva2:281137
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
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, 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.

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: 2017-12-14Bibliographically approved
3. Role of LPS in vesicle mediated export of Vibrio cholerae PrtV protease
Open this publication in new window or tab >>Role of LPS in vesicle mediated export of Vibrio cholerae PrtV protease
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Gram negative bacteria produce outer membrane vesicles (OMVs) during normal bacterial growth. Recent studies have shown that OMVs can transport biologically active toxins and enzymes to the environment and into the host. We have initiated analysis of OMV associated proteins in V. cholerae. In this study, V. cholerae wild type strain P27459 and the O-antigen ligase mutant waaL, which lacks the O-antigen of the LPS were analyzed for the OMV associated release of the secreted protease PrtV. OMVs from the waaL mutant showed a higher amount of associated secreted PrtV protein than the OMVs from wild type V. cholerae indicating that LPS might be involved in vesicle association of the PrtV protein. We showed that the OMV associated PrtV protein is biologically active.

National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-30256 (URN)
Available from: 2009-12-15 Created: 2009-12-15 Last updated: 2009-12-22Bibliographically approved
4. Evaluation of the presence of virulence-associated factors in Vibrio cholerae non-O1 non-O139 isolates from clinical and environmental sources
Open this publication in new window or tab >>Evaluation of the presence of virulence-associated factors in Vibrio cholerae non-O1 non-O139 isolates from clinical and environmental sources
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The broad group non-O1 non-O139 Vibrio cholerae presumably causes clinical diseases due to properties distinct from V. cholerae O1 and O139 serogroups in which cholera toxin is the hallmark of the infection. In this study, we screened for the presence of virulence-associated factors in V. cholerae non-O1 non-O139 strains isolated in Sweden. Six isolates from patients with different clinical manifestations and two environmental isolates were studied. None of the V. cholerae non-O1 non-O139 isolates carried the ctx or tcpA genes, but gene sequences for other putative virulence factors such as OmpU, cytolysin (VCC) and RTX were present. Significant differences in serum resistance were observed among the isolates independent of their encapsulation. The isolates were tested on Caenorhabditis elegans as an alternative host for analysis of factors associated with protection from natural predator grazing and environmental survival of V. cholerae. Three isolates caused lethality to C. elegans and also showed the strongest ability to resist killing by serum. We also observed that actin cross-linking activity and the level of protease secretion were different among the strains.

National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-30262 (URN)
Available from: 2009-12-15 Created: 2009-12-15 Last updated: 2009-12-22Bibliographically approved

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