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Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles
Umeå University, Faculty of Medicine, Department of Odontology.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (
Umeå University, Faculty of Medicine, Department of Odontology.
2013 (English)In: PLoS ONE, 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.

Place, publisher, year, edition, pages
2013. Vol. 8, no 1, e54661- p.
National Category
URN: urn:nbn:se:umu:diva-66687DOI: 10.1371/journal.pone.0054661PubMedID: 23382935OAI: diva2:608400
Available from: 2013-02-27 Created: 2013-02-27 Last updated: 2013-11-15Bibliographically 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. 56 p.
Umeå University odontological dissertations, ISSN 0345-7532 ; 129
Periodontitis, Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, membrane vesicles, vesicle-host cell interaction, protein delivery
National Category
Microbiology in the medical area
Research subject
Molecular Biology
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)
Available from: 2013-11-15 Created: 2013-11-11 Last updated: 2013-11-15Bibliographically approved

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Thay, BernardWai, Sun NyuntOscarsson, Jan
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