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NOD-like receptor activation by outer-membrane vesicles (OMVs) from non-O1 non-O139 Vibrio cholerae is modulated by the quorum sensing regulator HapR
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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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. Vol. 79, no 4, 1418-1427 p.
Keyword [en]
OMVs, peptidoglycan, NLR, CARD15, V. cholerae
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-39734DOI: 10.1128/IAI.00754-10PubMedID: 21263023OAI: oai:DiVA.org:umu-39734DiVA: diva2:402925
Available from: 2011-03-10 Created: 2011-02-07 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Outer membrane vesicle-mediated export of virulence factors from Gram-negative bacteria
Open this publication in new window or tab >>Outer membrane vesicle-mediated export of virulence factors from Gram-negative bacteria
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
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:nbn:se:umu:diva-57475 (URN)978-91-7459-451-5 (ISBN)
Public defence
2012-09-07, Astrid Fagraeussalen (A103UnodR1), By 6A, NUS, Umeå, 09:00 (English)
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Available from: 2012-08-17 Created: 2012-07-30 Last updated: 2012-08-17Bibliographically approved

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