Characterization Of Outer Membrane Vesicles Research Articles

Characterization of Outer Membrane Vesicles Produced by Vibrio vulnificus.

Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024.

Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1αandIL-1β. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

2nd Workshop in Microbial Molecular Biology

2nd Workshop in Microbial Molecular Biology

Outer Membrane Vesicles: Biogenesis, Functions, and Issues.

This review focuses on nonlytic outer membrane vesicles (OMVs), a subtype of bacterial extracellular vesicles (BEVs) produced by Gram-negative organisms focusing on the mechanisms of their biogenesis, cargo, and function. Throughout, we highlight issues concerning the characterization of OMVs and distinguishing them from other types of BEVs. We also highlight the shortcomings of commonly used methodologies for the study of BEVs that impact the interpretation of their functionality and suggest solutions to standardize protocols for OMV studies.

Validation of an FFF-MALS Method to Characterize the Production and Functionalization of Outer-Membrane Vesicles for Conjugate Vaccines.

With the ongoing development of conjugate vaccines battling infectious diseases, there is a need for novel carriers. Although tetanus toxoid and CRM197 belong to the traditional carrier proteins, outer-membrane vesicles (OMVs) are an excellent alternative: in addition to their size, OMVs have self-adjuvanting properties due to the presence of genetically detoxified lipopolysaccharide (LPS) and are therefore ideal as a vaccine component or antigen carrier. An essential aspect of their development for vaccine products is characterization of OMVs with respect to size and purity. We report on the development of a field-flow fractionation multiangle light-scattering (FFF-MALS) method for such characterization. Here, we introduced NIST-traceable particle-size standards and BSA as a model protein to verify the precision of the size and purity analysis of the OMVs. We executed a validation program according to the principles provided in the ICH Guidelines Q2 (R1) to assess the quality attributes of the results obtained by FFF-MALS analysis. All validation characteristics showed excellent results with coefficients of variation between 0.4 and 7.32%. Estimation of limits of detection for hydrodynamic radius and particle concentration revealed that as little as 1 μg OMV still yielded accurate results. With the validated method, we further characterized a full downstream purification process of our proprietary OMV. This was followed by the evaluation of other purified OMVs from different bacterial origin. Finally, functionalizing OMVs with N-γ-(maleimidobutyryl)oxysuccinimide-ester (GMBS), generating ready-to-conjugate OMVs, did not affect the structural integrity of the OMVs and as such, they could be evaluated with the validated FFF-MALS method.

Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692.

Pectobacterium brasiliense (Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria’s (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in Nicotiana benthamiana leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against Dickeya dadantii.

Characterization of Outer Membrane Vesicles from Fusobacterium nucleatum

Background/objectives: Fusobacterium nucleatum is an oral pathogen and is associated with the development of colorectal cancer (CRC). This study is to evaluate the ability of outer membrane vesicles (OMV) from F. nucleatum to modulate cellular responses in colonic cells. Methodology: Here we show that infection of colonic epithelial cells with F. nucleatum and its OMV induce pro-inflammatory chemokine and cytokine production and promote an EMT-like pheno- and genotypes in vitro as demonstrated by suppression of E-cadherin and up-regulation of several mesenchymal markers. F. nucleatum and its OMV modulate the barrier function of intestinal monolayers, a process likely related to their demonstrated ability to degrade E-cadherin and suppress its expression. Findings: Analysis of the OMV proteome by mass spectrometry demonstrates that they harbor the known virulence factors that appear to be enriched with proteolytic activity. Novelty/contribution: Taken together, these data indicate that F. nucleatum OMV have the potential to contribute to disease progression in the context of CRC. Keywords: Outer Membrane Vesicles, Fusobacterium nucleatum, Colorectal Cancer, Protease.

Proteomic characterization of outer membrane vesicles from gut mucosa-derived fusobacterium nucleatum

Fusobacterium nucleatum is a Gram-negative bacterium commonly found in the oral cavity and is often involved in periodontal diseases. Recent studies have shown increased F. nucleatum prevalence in colorectal cancer (CRC) tissues, and causal data has linked this bacterium to CRC tumorigenesis. Immune-based approaches to contain, reduce or eradicate its gut colonization may prevent CRC. Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria, typically contain multiple putative virulence factors and may elicit protective immune responses if used as vaccines. Here, OMVs were isolated from F. nucleatum cultures and purified using gradient centrifugation. Proteins contained within the OMVs were identified by nano LC/MS/MS analysis. Of 98 proteins consistently identified from duplicate analyses, 60 were predicted to localize to the outer membrane or periplasm via signal peptide driven translocation. Of these, six autotransporter proteins, which constitute the majority of protein mass of OMVs, were associated with Type V secretion system. In addition, other putative virulence factor proteins with functional domains, including FadA, MORN2 and YadA-like domain, were identified with multiple exposed epitope sites as determined by in silico analysis. Altogether, the non-replicative OMVs of F. nucleatum contain multiple antigenic virulence factors that may play important roles in the design and development of vaccines against F. nucleatum. SignificanceFusobacterium nulceatum has been proved playing significant role in colorectal carcinogenesis. Outer membrane vesicles are nanoparticles that naturally secreted by Gram-negative bacterial containing various antigenic components, which provides new insight in vaccine development. Understanding the constituents of F. nucleatum OMVs will provide fundamental information and potential strategies for OMV-based F. nucleatum vaccines design. Based on our knowledge this is the first proteomic study of OMVs from F. nucleatum.

Characterization and Immunogenicity of Outer Membrane Vesicles from Brucella abortus

Bovine brucellosis is a worldwide spread zoonotic disease. The objectives of this study were characterization of outer membrane vesicles from B. abortus and to evaluate their immunogenicity in mice. For this purpose, OMVs were derived from B. abortus strain 99 using ultracentrifugation method. Isolated OMVs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transmission electron microscopy which revealed spherical 20–300 nm structures rich in proteins. OMVs also showed immuno-reactivity with mice antisera in Western blot. Further, indirect ELISA showed specific and high-titer immune responses against the antigens present in OMVs suggesting their potential for a safe acellular vaccine candidate.

Characterization of outer membrane vesicles from a neonatal meningitic strain of Cronobacter sakazakii.

Cronobacter sakazakii is associated with severe and often fatal cases of infant meningitis and necrotizing enterocolitis. The form of meningitis differs from that due to Neisseria meningitidis and Streptococcus spp., in that it is highly invasive and destructive towards human brain cells. However, there is relatively little understanding of the cytopathogenic interaction of C. sakazakii with host cells which results in stimulation of an inflammatory immune response. The production of Cronobacter outer membrane vesicles (OMV) and their potential pathogenic functions have not yet been elucidated. This study is the first to show that C. sakazakii produce OMV, which may play a role in the activation of cytopathogenic and host cell responses on human intestinal epithelial cells. Cronobacter sakazakii strain 767 was used which had been isolated from a fatal outbreak of neonatal meningitis and necrotizing enterocolitis. Cronobacter sakazakii OMV were internalized by Caco-2 cells, increased cell proliferation and stimulated the host's innate proinflammatory response without inducing overt toxicity. A total of 18 OMV-associated proteins were identified by mass spectrometry and their potential pathogenicity roles were evaluated. Collectively, these data indicate that C. sakazakii OMV could play a role in pathogenesis by delivering bacterial toxins into host epithelial cells, driving proliferative and proinflammatory responses.

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis.

Identification of Outer Membrane Vesicles Derived from Orientia tsutsugamushi.

Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens.

Outer membrane vesicles of Pasteurella multocida contain virulence factors.

Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50–300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Ampr (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis.

Characterization of Outer Membrane Vesicles fromBrucella melitensisand Protection Induced in Mice

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3

Pseudoalteromonas antarctica NF3 is an Antarctic psychrotolerant Gram-negative bacterium that accumulates large amounts of an extracellular polymeric substance (EPS) with high protein content. Transmission electron microscopy analysis after high-pressure freezing and freeze substitution (HPF-FS) shows that the EPS is composed of a capsular polymer and large numbers of outer membrane vesicles (OMVs). These vesicles are bilayered structures and predominantly spherical in shape, with an average diameter of 25-70 nm, which is similar to what has been observed in OMVs from other Gram-negative bacteria. Analyses of lipopolysaccharide (LPS), phospholipids and protein profiles of OMVs are consistent with the bacterial outer membrane origin of these vesicles. In an initial attempt to elucidate the functions of OMVs proteins, we conducted a proteomic analysis on 1D SDS-PAGE bands. Those proteins putatively identified match with outer membrane proteins and proteins related to nutrient processing and transport in Gram-negative bacteria. This approach suggests that OMVs present in the EPS from P. antarctica NF3, might function to deliver proteins to the external media, and therefore play an important role in the survival of the bacterium in the extreme Antarctic environment.