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Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles are internalized in human host cells and trigger NOD1- and NOD2-dependent NF-κB activation
Umeå University, Faculty of Medicine, Department of Odontology. (Jan Oscarsson)ORCID iD: 0000-0001-5099-6844
University of Cologne. (Thomas Kufer)
University of Cologne. (Thomas Kufer)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Sun Nyunt Wai)
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2014 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 10, p. 4034-4046Article in journal (Refereed) Published
Abstract [en]

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis, and endocarditis. We recently demonstrated that OMVs disseminated by A. actinomycetemcomitans could deliver multiple proteins including biologically active cytolethal distending toxin (CDT) into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work we have used immunoelectron- and confocal microscopy analysis, and fluorescently labeled vesicles to further investigate mechanisms for A. actinomycetemcomitans OMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Co-localization analysis revealed that internalized OMVs co-localized with the endoplasmic reticulum, and carried antigens, detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role of A. actinomycetemcomitans OMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active PAMPs.

Place, publisher, year, edition, pages
American Society for Microbiology Press , 2014. Vol. 82, no 10, p. 4034-4046
National Category
Dentistry Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-92173DOI: 10.1128/IAI.01980-14ISI: 000341935100005PubMedID: 25024364OAI: oai:DiVA.org:umu-92173DiVA: diva2:739910
Note

Originally included in thesis in manuscript form.

Available from: 2014-08-22 Created: 2014-08-22 Last updated: 2018-02-13Bibliographically approved
In thesis
1. 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. p. 56
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)
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Supervisors
Available from: 2013-11-15 Created: 2013-11-11 Last updated: 2018-02-13Bibliographically approved

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