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

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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Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles

Toxins ◽

10.3390/toxins10100414 ◽

2018 ◽

Vol 10 (10) ◽

pp. 414 ◽

Cited By ~ 9

Author(s):

Justin Nice ◽

Nataliya Balashova ◽

Scott Kachlany ◽

Evan Koufos ◽

Eric Krueger ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Aggregatibacter Actinomycetemcomitans ◽

Aggressive Periodontitis ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Type I ◽

Outer Membranes ◽

Independent Mechanism ◽

Bacterial Outer Membrane

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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Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00937-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 19

Author(s):

Andreas Bauwens ◽

Lisa Kunsmann ◽

Helge Karch ◽

Alexander Mellmann ◽

Martina Bielaszewska

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Shiga Toxin ◽

Membrane Vesicles ◽

Polymyxin B ◽

Outer Membrane Vesicles ◽

Enterohemorrhagic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

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Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

Applied and Environmental Microbiology ◽

10.1128/aem.02567-17 ◽

2018 ◽

Vol 84 (8) ◽

pp. e02567-17 ◽

Cited By ~ 12

Author(s):

H. Bart van den Berg van Saparoea ◽

Diane Houben ◽

Marien I. de Jonge ◽

Wouter S. P. Jong ◽

Joen Luirink

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Salmonella Enterica ◽

Membrane Vesicles ◽

Therapeutic Agents ◽

Outer Membrane Vesicles ◽

High Density ◽

Content Type ◽

Protein Ligation ◽

Serovar Typhimurium

ABSTRACT The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

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Cholera Toxin Encapsulated within Several Vibrio cholerae O1 Serotype Inaba Outer Membrane Vesicles Lacks a Functional B-Subunit

Toxins ◽

10.3390/toxins11040207 ◽

2019 ◽

Vol 11 (4) ◽

pp. 207 ◽

Cited By ~ 2

Author(s):

Elnaz Rasti ◽

Angela Brown

Keyword(s):

Outer Membrane ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Host Cells ◽

Free Form ◽

Type Ii Secretion ◽

Secondary Mechanism

Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.

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Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Applied and Environmental Microbiology ◽

10.1128/aem.01941-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5854-5865 ◽

Cited By ~ 41

Author(s):

Maria H. Daleke-Schermerhorn ◽

Tristan Felix ◽

Zora Soprova ◽

Corinne M. ten Hagen-Jongman ◽

David Vikström ◽

...

Keyword(s):

Immune Responses ◽

Outer Membrane ◽

Vaccine Development ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Heterologous Proteins ◽

Content Type ◽

Serovar Typhimurium ◽

Heterologous Antigens

ABSTRACTOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of theMycobacterium tuberculosisantigens ESAT6, Ag85B, and Rv2660c were targeted to the surface ofEscherichia coliOMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolRΔtolAderivative of attenuatedSalmonella entericaserovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by theM. tuberculosisantigens and epitopes fromChlamydia trachomatismajor outer membrane protein (MOMP). Also, we showed that delivery ofSalmonellaOMVs displaying Ag85B to antigen-presenting cellsin vitroresults in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

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Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium

Applied and Environmental Microbiology ◽

10.1128/aem.00829-19 ◽

2019 ◽

Vol 85 (19) ◽

Cited By ~ 2

Author(s):

Tanja Fischer ◽

Martin Schorb ◽

Greta Reintjes ◽

Androniki Kolovou ◽

Rachel Santarella-Mellwig ◽

...

Keyword(s):

Outer Membrane ◽

Surface Area ◽

Algal Blooms ◽

Membrane Vesicle ◽

Outer Membrane Proteins ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Glycoside Hydrolases ◽

Content Type ◽

Degradative Enzymes

ABSTRACT Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 μm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms. IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

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Cytotoxic Necrotizing Factor Type 1 Delivered by Outer Membrane Vesicles of Uropathogenic Escherichia coli Attenuates Polymorphonuclear Leukocyte Antimicrobial Activity and Chemotaxis

Infection and Immunity ◽

10.1128/iai.00637-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4401-4408 ◽

Cited By ~ 54

Author(s):

Jon M. Davis ◽

Humberto M. Carvalho ◽

Susan B. Rasmussen ◽

Alison D. O'Brien

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Single Amino Acid ◽

Upec Strain ◽

Cytotoxic Necrotizing Factor ◽

Factor Type

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1), a toxin produced by many strains of uropathogenic Escherichia coli (UPEC), constitutively activates small GTPases of the Rho family by deamidating a single amino acid within these target proteins. Such activated GTPases not only stimulate actin polymerization within affected cells but also, as we previously reported, decrease membrane fluidity on mouse polymorphonuclear leukocytes (PMNs). In that same investigation we found that this diminished membrane movement impedes the clustering of the complement receptor CD11b/CD18 on PMNs and, in turn, decreases PMN phagocytic capacity and microbicidal activity on PMNs in direct contact with CNF1-expressing UPEC as well as on those in proximity to wild-type UPEC. The latter observation suggested to us that CNF1 is released from neighboring bacteria, although at the time of initiation of the study described here, no specific mechanism for export of CNF1 from UPEC had been described. Here we present evidence that CNF1 is released from the CNF1-expressing UPEC strain CP9 (serotype O4/H5/K54) in a complex with outer membrane vesicles (OMVs) and that these CNF1-bearing vesicles transfer biologically active CNF1 to PMNs and attenuate phagocyte function. Furthermore, we show that CNF1-bearing vesicles act in a dose-dependent fashion on PMNs to inhibit their chemotactic response to formyl-Met-Leu-Phe, while purified CNF1 does not. We conclude that OMVs provide a means for delivery of CNF1 from a UPEC strain to PMNs and thus negatively affect the efficacy of the acute inflammatory response to these organisms.

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Horizontal Transfer of the OXA-24 Carbapenemase Gene via Outer Membrane Vesicles: a New Mechanism of Dissemination of Carbapenem Resistance Genes in Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00929-10 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3084-3090 ◽

Cited By ~ 134

Author(s):

Carlos Rumbo ◽

Esteban Fernández-Moreira ◽

María Merino ◽

Margarita Poza ◽

Jose Antonio Mendez ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Resistance Genes ◽

Carbapenem Resistance ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Content Type ◽

Class D ◽

Carbapenemase Gene

ABSTRACTThe resistance ofAcinetobacter baumanniistrains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of β-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes amongA. baumanniistrains are not fully understood. In this study we used two carbapenem-resistant clinical strains ofA. baumannii(AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borneblaOXA-24gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate thatA. baumanniireleases outer membrane vesicles (OMVs) duringin vitrogrowth. The use of hybridization studies enabled us to show that these OMVs harbored theblaOXA-24gene. The incubation of these OMVs with the carbapenem-susceptibleA. baumanniiATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring theblaOXA-24gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboringblaOXA-24were released byA. baumanniiATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates ofA. baumanniimay release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surroundingA. baumanniibacterial isolates.

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