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Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin (CDT) from aggregatibacter actinomycetemcomitans
Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Wai)
Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Charpentier)
Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
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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. Vol. 80, no 1, 31-42 p.
National Category
Dentistry
Identifiers
URN: urn:nbn:se:umu:diva-49740DOI: 10.1128/IAI.06069-11PubMedID: 22025516OAI: oai:DiVA.org:umu-49740DiVA: diva2:456972
Available from: 2011-11-16 Created: 2011-11-16 Last updated: 2017-12-08Bibliographically 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)
Opponent
Supervisors
Available from: 2012-08-17 Created: 2012-07-30 Last updated: 2012-08-17Bibliographically approved
2. Vesicle-mediated and free soluble delivery of bacterial effector proteins by oral and systemic pathogens
Open this publication in new window or tab >>Vesicle-mediated and free soluble delivery of bacterial effector proteins by oral and systemic pathogens
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus.

Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form.

We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, 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. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells.

Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2013. 56 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 129
Keyword
Periodontitis, Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, membrane vesicles, vesicle-host cell interaction, protein delivery
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-82782 (URN)978-91-7459-751-6 (ISBN)
Public defence
2013-12-06, Sal D, Plan 9, Tändläkarhögskolan, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-11-15 Created: 2013-11-11 Last updated: 2013-11-15Bibliographically approved

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