Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

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Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

Infection and Immunity ◽

10.1128/iai.06069-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 31-42 ◽

Cited By ~ 61

Author(s):

Pramod Kumar Rompikuntal ◽

Bernard Thay ◽

Muhammad Khanzeb Khan ◽

Jonna Alanko ◽

Anna-Maija Penttinen ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Cell Types ◽

Aggregatibacter Actinomycetemcomitans ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Cytolethal Distending Toxin ◽

Content Type ◽

Serotype A ◽

Tight Association

ABSTRACTAggregatibacter actinomycetemcomitansis implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identifiedA. actinomycetemcomitansouter 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 wholeA. actinomycetemcomitansserotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated fromA. actinomycetemcomitansstrain D7SS (serotype a), in contrast to OMVs from a D7SScdtABCmutant, 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 inA. actinomycetemcomitansisolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved inA. actinomycetemcomitans. Although the role ofA. actinomycetemcomitansOMVs 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.

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Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

Infection and Immunity ◽

10.1128/iai.01980-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4034-4046 ◽

Cited By ~ 59

Author(s):

Bernard Thay ◽

Anna Damm ◽

Thomas A. Kufer ◽

Sun Nyunt Wai ◽

Jan Oscarsson

Keyword(s):

Outer Membrane ◽

Hela Cells ◽

Inflammatory Responses ◽

Membrane Vesicles ◽

Aggregatibacter Actinomycetemcomitans ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Host Cells ◽

Content Type ◽

Human Embryonic Kidney Cells

ABSTRACTAggregatibacter actinomycetemcomitansis an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. We recently demonstrated that outer membrane vesicles (OMVs) disseminated byA. actinomycetemcomitanscould 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 forA. actinomycetemcomitansOMV-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. Colocalization analysis revealed that internalized OMVs colocalized with the endoplasmic reticulum and carried antigens, detected using an antibody specific to wholeA. actinomycetemcomitansserotype 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 ofA. actinomycetemcomitansOMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs).

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Ultrastructural Analysis of Membrane Morphology and Outer Membrane Vesicles Associated with Aggregatibacter actinomycetemcomitans

Microscopy and Microanalysis ◽

10.1017/s1431927612002267 ◽

2012 ◽

Vol 18 (S2) ◽

pp. 82-83

Author(s):

F. Azari ◽

L. Nyland ◽

T. Ruiz ◽

C. Yu ◽

K. Mintz

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Aggregatibacter Actinomycetemcomitans ◽

Outer Membrane Vesicles ◽

Ultrastructural Analysis ◽

Membrane Morphology

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

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The Effect of Helicobacter Pylori Outer Membrane Vesicles On Vacuolation in Gastric and Colonic Epithelial Cells

Endoscopy ◽

10.1055/s-2006-956848 ◽

2006 ◽

Vol 38 (11) ◽

Author(s):

EM Mullaney ◽

AM Terres ◽

DP Kelleher

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Colonic Epithelial Cells

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Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

10.26434/chemrxiv.8282204.v1 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Rémi Terrasse ◽

Jayesh Arun Bafna ◽

Lorraine Benier ◽

Mathias Winterhalter

Keyword(s):

Outer Membrane ◽

Lipid Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Multi Drug Resistance ◽

Gram Negative Bacteria ◽

Membrane Channels ◽

Gram Negative ◽

Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from Escherichia coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (kon) and lower dissociation (koff) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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