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Outer membrane vesicle-mediated export of virulence factors from Gram-negative bacteria
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Sun Nyunt Wai)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2012. , 55 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1512
Keyword [en]
Outer membrane vesicles, CDT, PGN, PrtV, Campylobacter jejuni, Vibrio cholerae, Aggregatibacter actinomycetemcomitans
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-57475ISBN: 978-91-7459-451-5 (print)OAI: oai:DiVA.org:umu-57475DiVA: diva2:542664
Public defence
2012-09-07, Astrid Fagraeussalen (A103UnodR1), By 6A, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-08-17 Created: 2012-07-30 Last updated: 2012-08-17Bibliographically approved
List of papers
1. Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni
Open this publication in new window or tab >>Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni
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2009 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 16, no 9, 220- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.

RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.

CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-31620 (URN)10.1186/1471-2180-9-220 (DOI)19835618 (PubMedID)
Available from: 2010-02-11 Created: 2010-02-11 Last updated: 2017-12-12Bibliographically approved
2. NOD-like receptor activation by outer-membrane vesicles (OMVs) from non-O1 non-O139 Vibrio cholerae is modulated by the quorum sensing regulator HapR
Open this publication in new window or tab >>NOD-like receptor activation by outer-membrane vesicles (OMVs) from non-O1 non-O139 Vibrio cholerae is modulated by the quorum sensing regulator HapR
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2011 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, no 4, 1418-1427 p.Article in journal (Refereed) Published
Abstract [en]

Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. siRNA-mediated gene knock-down revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain, leucine rich repeat containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum sensing regulator HapR which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs in infective conditions. This work thus identified a new facet of how Vibrio affects host immune responses and defines a role for the quorum sensing machinery in this process.

Place, publisher, year, edition, pages
American Society for Microbiology, 2011
Keyword
OMVs, peptidoglycan, NLR, CARD15, V. cholerae
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-39734 (URN)10.1128/IAI.00754-10 (DOI)21263023 (PubMedID)
Available from: 2011-03-10 Created: 2011-02-07 Last updated: 2017-12-11Bibliographically approved
3. Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin (CDT) from aggregatibacter actinomycetemcomitans
Open this publication in new window or tab >>Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin (CDT) from aggregatibacter actinomycetemcomitans
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2012 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 80, no 1, 31-42 p.Article in journal (Refereed) Published
Abstract [en]

Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similar to several other Gram-negative species this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G(2) cell cycle arrest and/or apoptosis in many mammalian cell types. In this study we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a) in contrast to OMVs from a D7SS cdtABC mutant induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates, belonging to serotypes b, and c, respectively, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although, the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.

Place, publisher, year, edition, pages
American Society for Microbiology, 2012
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-49740 (URN)10.1128/IAI.06069-11 (DOI)22025516 (PubMedID)
Available from: 2011-11-16 Created: 2011-11-16 Last updated: 2017-12-08Bibliographically approved
4. Outer membrane vesicle-mediated export of PrtV protease from Vibrio cholerae
Open this publication in new window or tab >>Outer membrane vesicle-mediated export of PrtV protease from Vibrio cholerae
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Gram-negative bacteria release large amounts of outer membrane vesicles (OMVs) during normal growth. OMVs from pathogenic bacteria are known to carry different biologically active toxins and enzymes into the surrounding environment. We hypothesized that OMVs may therefore be able to mediate the transport of bacterial products into host cells. We present here an analysis of the V. cholerae OMV-associated virulence factor PrtV. 

Methodology/Principal Findings: We observed that PrtV, a M6 family, zinc-binding metalloprotease, is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs. The association of PrtV with OMVs was determined by immunoblotting and electron microscopy using immunogold labeling. In addition, we observed that the PKD-domain(s) of PrtV has a role in the protein’s association with OMVs. We also demonstrated that OMV-associated PrtV was biologically active because HCT8 cells treated with OMVs from the wild type V. cholerae strain C6706 exhibited altered morphology, whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Moreover, our data suggest that OMV-associated PrtV might be transported into target eukaryotic cells by a vesicle fusion mechanism in association with lipid raft microdomains in the plasma membrane.

Conclusion/Significance: Our findings suggest that OMVs can deliver biologically active PrtV into target host cells.

National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-57474 (URN)
Available from: 2012-07-30 Created: 2012-07-30 Last updated: 2012-08-27Bibliographically approved

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