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Vesicle-mediated and free soluble delivery of bacterial effector proteins by oral and systemic pathogens
Umeå University, Faculty of Medicine, Department of Odontology. (Jan Oscarsson)ORCID iD: 25936794100
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 [en]
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: urn:nbn:se:umu:diva-82782ISBN: 978-91-7459-751-6 (print)OAI: oai:DiVA.org:umu-82782DiVA: diva2:663252
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
List of papers
1. Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells
Open this publication in new window or tab >>Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells
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2008 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, no 206, 13- p.Article in journal (Refereed) Published
Abstract [en]

BackgroundAggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells.

Results: By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.

ConclusionA. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.

Place, publisher, year, edition, pages
London: BioMed Central, 2008
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-31882 (URN)10.1186/1471-2180-8-206 (DOI)19038023 (PubMedID)
Available from: 2010-02-21 Created: 2010-02-21 Last updated: 2017-12-12Bibliographically approved
2. 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
3. Aggregatibacter actinomycetemcomitans outer membrane vesicles are internalized in human host cells and trigger NOD1- and NOD2-dependent NF-kB activation
Open this publication in new window or tab >>Aggregatibacter actinomycetemcomitans outer membrane vesicles are internalized in human host cells and trigger NOD1- and NOD2-dependent NF-kB activation
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-82981 (URN)
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2013-11-15Bibliographically approved
4. Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles
Open this publication in new window or tab >>Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1, e54661- p.Article in journal (Refereed) Published
Abstract [en]

Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla) to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-66687 (URN)10.1371/journal.pone.0054661 (DOI)23382935 (PubMedID)
Available from: 2013-02-27 Created: 2013-02-27 Last updated: 2017-12-06Bibliographically approved

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