scholarly journals Context-Dependent Activation Kinetics Elicited by Soluble versus Outer Membrane Vesicle-Associated Heat-Labile Enterotoxin

2011 ◽  
Vol 79 (9) ◽  
pp. 3760-3769 ◽  
Author(s):  
Halima Chutkan ◽  
Meta J. Kuehn
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Extracellular Vesicle ◽  
Outer Membrane Vesicles ◽  
Cytokine Gene Expression ◽  
Activator Protein 1 ◽  
Content Type ◽  
Creb Phosphorylation ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is the leading cause of traveler's diarrhea and children's diarrhea worldwide. Among its virulence factors, ETEC produces heat-labile enterotoxin (LT). Most secreted LT is associated with outer membrane vesicles that are rich in lipopolysaccharide. The majority of prior studies have focused on soluble LT purified from ETEC periplasm. We investigated the hypothesis that the extracellular vesicle context of toxin presentation might be important in eliciting immune responses. We compared the polarized epithelial cell responses to apically applied soluble LT and LT-containing vesicles (LT+vesicles) as well as controls using a catalytically inactive mutant of LT and vesicles lacking LT. Although vesicle treatments with no or catalytically inactive LT induced a modest amount of interleukin-6 (IL-6), samples containing catalytically active LT elicited higher levels. A combination of soluble LT and LT-deficient vesicles induced significantly higher IL-6 levels than either LT or LT+vesicles alone. The responses to LT+vesicles were found to be independent of the canonical LT pathway, because the inhibition of cyclic AMP response element (CRE)-binding protein (CREB) phosphorylation did not lead to a decrease in cytokine gene expression levels. Furthermore, soluble LT caused earlier phosphorylation of CREB and activation of CRE compared with LT+vesicles. Soluble LT also led to the activation of activator protein 1, whereas LT+vesicle IL-6 responses appeared to be mediated by NF-κB. In summary, the results demonstrate that soluble LT and vesicle-bound LT elicit ultimately similar cytokine responses through distinct different activation pathways.

scholarly journals Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium

2019 ◽  
Vol 85 (19) ◽  
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.

scholarly journals New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

2013 ◽  
Vol 79 (6) ◽  
pp. 1874-1881 ◽  
Author(s):  
Carla Pérez-Cruz ◽  
Ornella Carrión ◽  
Lidia Delgado ◽  
Gemma Martinez ◽  
Carmen López-Iglesias ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Dna Content ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Freeze Substitution ◽  
Outer Membrane Vesicles ◽  
Negative Bacterium ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type

ABSTRACTOuter membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacteriumShewanella vesiculosaM7Thas revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacteriumShewanella vesiculosaM7Tthat can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).

scholarly journals Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

2011 ◽  
Vol 18 (11) ◽  
pp. 1803-1808 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
George P. Munson ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protective Efficacy ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Virulence Proteins ◽  
Protective Antigens ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines.

scholarly journals Pseudomonas Quinolone Signal-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Adam C. Cooke ◽  
Catalina Florez ◽  
Elise B. Dunshee ◽  
Avery D. Lieber ◽  
Michelle L. Terry ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Burn Wound ◽  
Content Type ◽  
Enhanced Production ◽  
Biofilm Dispersion ◽  
Biofilm Development

ABSTRACT Bacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of antibiofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital-acquired infections and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-cell communication, virulence factor delivery, and transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Several groups have shown that Pseudomonas quinolone signal (PQS) induces OMV formation in P. aeruginosa. Our group described a biophysical mechanism for this and recently showed it is operative in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype was rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components. IMPORTANCE Treatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. Pseudomonas quinolone signal (PQS) has been implicated in early biofilm development, but we hypothesized that its function as an outer membrane vesicle (OMV) inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and provide evidence that effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. These findings lay the groundwork for understanding OMV contributions to biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.

scholarly journals Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis tolB Mutant

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Kotaro Takaki ◽  
Yuhei O. Tahara ◽  
Nao Nakamichi ◽  
Yusuke Hasegawa ◽  
Masaki Shintani ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Striking Similarity ◽  
Common Mechanism ◽  
Separation And Purification ◽  
Intercellular Interaction ◽  
Electron Microscopic ◽  
Content Type

ABSTRACT Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles. IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090T ΔtolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.

scholarly journals Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1661
Author(s):  
Mei-Hsiu Chen ◽  
Tse-Ying Liu ◽  
Yu-Chiao Chen ◽  
Ming-Hong Chen
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicle ◽  
Glioma Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
E Coli ◽  
Tumor Bearing ◽  
Nano Gold ◽  
Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

scholarly journals A Critical Threshold of Meningococcal Factor H Binding Protein Expression Is Required for Increased Breadth of Protective Antibodies Elicited by Native Outer Membrane Vesicle Vaccines

2011 ◽  
Vol 18 (5) ◽  
pp. 736-742 ◽  
Author(s):  
Oliver Koeberling ◽  
Isabel Delany ◽  
Dan M. Granoff
Keyword(s):  
Outer Membrane ◽  
Binding Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Factor H ◽  
Critical Threshold ◽  
Recombinant Strains ◽  
Endotoxin Activity ◽  
Group B

ABSTRACTNative outer membrane vesicles (NOMV) (not detergent treated), which are prepared from recombinant strains with attenuated endotoxin activity and overexpressed factor H binding protein (fHbp), elicited broad serum bactericidal antibody responses in mice. The amount of overexpressed fHbp required for optimal immunogenicity is not known. In this study we prepared NOMV vaccines from LpxL1 knockout (ΔLpxL1) mutants with penta-acylated lipooligosaccharide and attenuated endotoxin activity. The recombinant strains had wild-type (1×) fHbp expression or were engineered for 3-fold- or 10-fold-increased fHbp expression (3× or 10× fHbp). Control vaccines included NOMV from ΔLpxL1/ΔfHbp mutants or recombinant fHbp. In mice, only the 10× fHbp NOMV vaccine elicited significantly higher serum IgG anti-fHbp antibody titers than the corresponding 1× fHbp NOMV or recombinant fHbp vaccine. The 10× fHbp NOMV vaccine also elicited higher bactericidal responses (P< 0.05) against five group B strains with heterologous PorA than the recombinant fHbp or 1× fHbp NOMV vaccine. The 3× fHbp NOMV vaccine gave higher bactericidal titers against only one strain. Serum bactericidal titers in mice immunized with the control ΔfHbp NOMV vaccines were <1:10, and bactericidal titers in mice immunized with the 10× fHbp NOMV vaccine were <1:10 after adsorption of anti-fHbp antibodies. Mixing antiserum to NOMV vaccines from fHbp knockout mutants with antiserum to recombinant fHbp did not increase anti-fHbp bactericidal titers. Thus, a critical threshold of increased fHbp expression is required for NOMV vaccines to elicit broad serum bactericidal responses, and the antibodies conferring protection are directed primarily at fHbp.

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

2017 ◽  
Vol 61 (9) ◽  
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.

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

2018 ◽  
Vol 84 (8) ◽  
pp. e02567-17 ◽  
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.

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

2014 ◽  
Vol 80 (18) ◽  
pp. 5854-5865 ◽  
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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