Search Results (24)

Outer membrane vesicles (OMVs) are tractable, cell-free microbial outputs that can shape innate immune programs. Here, we compared OMVs from the probiotic Escherichia coli Nissle 1917 (EcN) and the commensal strain ECOR12 in a paired within-donor model of human monocyte-derived dendritic cells (Mo-DCs) (N = 20). In the core integrated arm, Mo-DCs were exposed to iDC control, EcN OMVs, or ECOR12 OMVs (10 µg/mL, 24 h) and profiled for maturation markers (CD14, CD83, CD209), cytokines (IL-6, TNF-α, IL-10), and a targeted miRNA panel (miR-155-5p, let-7i-3p, miR-146b-5p, miR-29a-5p). Both OMV types promoted maturation (increased CD83 and reduced CD14), but generated distinct cytokine–miRNA configurations, with ECOR12 tending toward an IL-10–high profile and EcN toward higher IL-6/TNF-α tendencies. Multivariate integration separated conditions into reproducible, strain-specific immune fingerprints, supporting the key take-home that probiotic versus commensal E. coli OMVs imprint distinguishable coordinated response states in human DCs. In an extended phenotyping arm, ECOR63 OMVs were evaluated by ELISA and flow cytometry only and were not included in miRNA profiling or integrated PCA due to unavailable matched miRNA measurements. Full article

Background/Objectives: Bacterial outer membrane vesicles (OMVs) play a role in bacterial communication, virulence, antimicrobial resistance, and host–pathogen interaction. OMV isolation is a key step for studying these particles’ functions; nevertheless, isolation procedures can greatly influence the yield, purity, and structural integrity of OMVs, thereby affecting downstream biological analyses and functional interpretation. Methods: In this study, we compared the efficacy of two OMV isolation techniques, differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC), in separating and concentrating vesicles produced by two Escherichia coli strains belonging to uropathogenic (UPEC) and Shiga toxin-producing (STEC) pathotypes. The isolated OMVs were characterized using a multi-analytical approach including transmission and scanning electron microscopy (TEM, SEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), ζ-potential measurement, and protein quantification to assess the purity of the preparations. Results: Samples obtained by dUC exhibited higher total protein content, broader particle size distributions, and more pronounced contamination by non-vesicular material. In contrast, SEC yielded morphologically homogeneous and structurally well-preserved vesicles, higher particle-to-protein ratios, and lower total protein content, reflecting reduced co-isolation of protein aggregates. NTA and DLS analyses revealed polydisperse populations in samples obtained with both isolation methods, with DLS measurements highlighting the contribution of larger or transient aggregates. ζ-potential values were close to neutrality for all samples, consistent with limited electrostatic repulsion and with the aggregation tendencies observed in some preparations. Conclusions: This study describes features of OMV produced by two relevant E. coli strains considering two isolation strategies which exert method- and strain-dependent effects on vesicle properties, including size distribution and surface charge, and emphasizes the trade-offs between yield, purity, and vesicle integrity. Full article

The global rise of antimicrobial resistance represents a critical challenge to public health, with Escherichia coli emerging as one of the most significant contributors due to its high adaptability and prevalence of extended-spectrum β-lactamase (ESBL) production. Outer membrane vesicles (OMVs), nanoscale structures released by Gram-negative bacteria, have recently been implicated in the dissemination of resistance determinants and direct antibiotic inactivation. This study investigated the role of OMVs derived from ESBL-producing E. coli in mediating resistance to ampicillin. Clinical strains harboring CTX-M-15 resistance genes were cultured under selective pressure, and OMVs were purified via size-exclusion chromatography. Characterization using tunable resistive pulse sensing (TRPS) and cryo-transmission electron microscopy confirmed vesicle integrity, with sizes ranging from 80 to 150 nm. DNA quantification and PCR analysis revealed the presence of CTX-M-15 genes within vesicles, which remained protected from DNase digestion, confirming encapsulation. Functional assays demonstrated β-lactamase activity within OMVs, with proteinase K treatment indicating localization primarily within vesicles rather than on their surface. Importantly, OMVs inactivated ampicillin in a dose-dependent manner, significantly reducing its efficacy against susceptible E. coli. Disc diffusion and microtiter plate assays confirmed that β-lactamase-positive OMVs protected susceptible strains from antibiotic killing, promoting bacterial survival and growth. This study uniquely demonstrates that OMVs from CTX-M-15–producing Escherichia coli carry both resistance genes and active β-lactamase enzymes, thereby facilitating both genetic dissemination and direct antibiotic inactivation. Targeting OMV biogenesis may represent a novel strategy to combat antimicrobial resistance. Full article

Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings. Full article

Escherichia coli (E. coli) is commonly utilized as a vehicle for anti-tumor therapy due to its unique tumor-targeting capabilities and ease of engineering modification. To further explore the role of E. coli in tumor treatment, we consider that E. coli outer membrane vesicles (E. coli-OMVs) play a crucial role in the therapeutic process. Firstly, E. coli-OMVs were isolated and partially purified by filtration and ultracentrifugation, and were characterized using techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western Blot (WB). The obtained extracellular nanoparticles, containing OMVs, were found to inhibited the growth of CT26 tumor in mice, while the expression of Bax protein was increased and the expression of Bcl-2 protein decreased. In vitro experiments showed that E. coli-OMVs entered CT26 cells and inhibited cell proliferation, invasion and migration. In addition, in the presence of E. coli-OMVs, we observed an increase in apoptosis rate and a decrease in the ratio of Bcl-2/Bax. These data indicate that E. coli-OMVs inhibits the growth of CT26 colon cancer by inducing apoptosis of CT26 cells. These findings propose E. coli-OMVs as a promising therapeutic drug for colorectal cancer (CRC), providing robust support for further research in related fields. Full article

Bacterial extracellular vesicles (bEVs) secreted by Gram-negative bacteria are referred to as outer membrane vesicles (OMVs) because they originate in the outer membrane. OMVs are membrane-coated vesicles 20–250 nm in size. They contain lipopolysaccharide (LPS), peptidoglycan, proteins, lipids, nucleic acids, and other substances derived from their parent bacteria and participate in the transmission of information to host cells. OMVs have broad prospects in terms of potential application in the fields of adjuvants, vaccines, and drug delivery vehicles. Currently, there remains a lack of efficient and convenient methods to isolate OMVs, which greatly limits OMV-related research. In this study, we developed a fast, convenient, and low-cost gradient filtration method to separate OMVs that can achieve industrial-scale production while maintaining the biological activity of the isolated OMVs. We compared the gradient filtration method with traditional ultracentrifugation to isolate OMVs from probiotic Escherichia coli Nissle 1917 (EcN) bacteria. Then, we used RAW264.7 macrophages as an in vitro model to study the influence on the immune function of EcN-derived OMVs obtained through the gradient filtration method. Our results indicated that EcN-derived OMVs were efficiently isolated using our gradient filtration method. The level of OMV enrichment obtained via our gradient filtration method was about twice as efficient as that achieved through traditional ultracentrifugation. The EcN-derived OMVs enriched through the gradient filtration method were successfully taken up by RAW264.7 macrophages and induced them to secrete pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukins (ILs) 6 and 1β, as well as anti-inflammatory cytokine IL-10. Furthermore, EcN-derived OMVs induced more anti-inflammatory response (i.e., IL-10) than pro-inflammatory response (i.e., TNF-α, IL-6, and IL-1β). These results were consistent with those reported in the literature. The related literature reported that EcN-derived OMVs obtained through ultracentrifugation could induce stronger anti-inflammatory responses than pro-inflammatory responses in RAW264.7 macrophages. Our simple and novel separation method may therefore have promising prospects in terms of applications involving the study of OMVs. Full article

Antimicrobial resistance (AMR) has been recognized as one of the most important crises affecting global human health in the 21st century. Tigecycline is one of the last resort antibiotics for treating severe infections caused by multi-drug resistant Enterobacteriaceae. However, the mobile resistance gene tet(X4), which could mediate high-level tigecycline resistance, was discovered in 2019. The outer membrane vesicle (OMV) has been recognized as a new route for horizontal gene transfer; antimicrobial resistant bacteria also have the ability to secret OMVs, while little is known about the impact of antibiotics on the secretion and characteristics of OMVs from tigecycline resistant bacteria till now. This study aimed to investigate the effects of antibiotics on the production and traits of a tigecycline resistant Escherichia coli strain of 47EC. The results showed that sub-inhibitory (1/2 MIC or 1/4 MIC) concentrations of gentamicin, meropenem, ceftazidime, chloramphenicol, tigecycline, ciprofloxacin, polymycin, rifaximin and mitomycin C could significantly increase the secretion of OMVs (0.713 ± 0.05~6.333 ± 0.15 mg/mL) from E. coli 47EC compared to the respective untreated control (0.709 ± 0.03 mg/mL). In addition, the particle sizes of OMVs were generally larger, and the zeta potential were lower in the antibiotics-treated groups than those of the antibiotic-free group. The copy numbers of the tigecycline resistance gene of tet(X4) in the OMVs of most antimicrobial-treated groups were higher than that of the control group. Moreover, transcriptome analysis on ciprofloxacin-treated E. coli 47EC indicated that the SOS response and prophage activation might participate in the ciprofloxacin-induced OMV formation. In conclusion, the clinical application of antibiotics in treating bacterial infections, especially multi-drug resistant bacteria, might lead to the increased secretion of bacterial OMVs and the enrichment of antimicrobial-resistant genes in the OMVs. Full article

A cell’s ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in E. coli REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host. Full article

Human papillomaviruses (HPVs) are a large family of viruses with a capsid composed of the L1 and L2 proteins, which bind to receptors of the basal epithelial cells and promote virus entry. The majority of sexually active people become exposed to HPV and the virus is the most common cause of cervical cancer. Vaccines are available based on the L1 protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted and their costs limit broad vaccination campaigns. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51, and 59 were shown to elicit broadly neutralizing anti-HPV antibodies. In this study, we tested whether E. coli outer membrane vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform owing to their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide composed of the L2 epitopes from serotypes 18, 33, 35, and 59 provided a broad cross-protective activity against a large panel of HPV serotypes as determined using pseudovirus neutralization assay. Considering the simplicity of the OMV production process, our work provides a highly effective and inexpensive solution to produce universal anti-HPV vaccines. Full article

In situ vaccination (ISV) is a promising cancer immunotherapy strategy that consists of the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site, and therefore, to elicit an effective anti-tumor immune response, all that is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial outer membrane vesicles (OMVs) are potent adjuvants since they contain several microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work, we first show that the OMVs from E. coli BL21(DE3)Δ60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of three different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse model. Overall, our data support the use of the OMVs in ISV and indicate that ISV efficacy can benefit from the addition of properly selected tumor-specific neo-antigens. Full article

All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein “anchors/directors”) and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp’, SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp’. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp’ anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs. Full article

Chlamydia trachomatis is the bacterial pathogen that causes most cases of sexually transmitted diseases annually. To combat the global spread of asymptomatic infection, development of effective (mucosal) vaccines that offer both systemic and local immune responses is considered a high priority. In this study, we explored the expression of C. trachomatis full-length (FL) PmpD, as well as truncated PmpD passenger constructs fused to a “display” autotransporter (AT) hemoglobin protease (HbpD) and studied their inclusion into outer membrane vesicles (OMVs) of Escherichia coli and Salmonella Typhimurium. OMVs are considered safe vaccine vectors well-suited for mucosal delivery. By using E. coli AT HbpD-fusions of chimeric constructs we improved surface display and successfully generated Salmonella OMVs decorated with a secreted and immunogenic PmpD passenger fragment (aa68-629) to 13% of the total protein content. Next, we investigated whether a similar chimeric surface display strategy could be applied to other AT antigens, i.e., secreted fragments of Prn (aa35-350) of Bordetella pertussis and VacA (aa65-377) of Helicobacter pylori. The data provided information on the complexity of heterologous expression of AT antigens at the OMV surface and suggested that optimal expression strategies should be developed on an antigen-to-antigen basis. Full article

Extracellular vesicles (EVs) are critical elements of cell–cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation. Full article

Escherichia coli releases outer membrane vesicles (OMVs) into the extracellular environment. OMVs, which contain the outer membrane protein, lipopolysaccharides (LPS), and genetic material, play an important role in immune response modulation. An isobaric tag for relative and absolute quantitation (iTRAQ) analysis was used to investigate OMV constituent proteins and their functions in burn trauma. OMV sizes ranged from 50 to 200 nm. Proteomics and Gene Ontology analysis revealed that ΔrfaC and ΔrfaG were likely involved in the upregulation of the structural constituent of ribosomes for the outer membrane and of proteins involved in protein binding and OMV synthesis. ΔrfaL was likely implicated in the downregulation of the structural constituent of the ribosome, translation, and cytosolic large ribosomal subunit. Kyoto Encyclopedia of Genes and Genomes analysis indicated that ΔrfaC and ΔrfaG downregulated ACP, ACEF, and ADHE genes; ΔrfaL upregulated ACP, ACEF, and ADHE genes. Heat map analysis demonstrated upregulation of galF, clpX, accA, fabB, and grpE and downregulation of pspA, ydiY, rpsT, and rpmB. These results suggest that RfaC, RfaG, and RfaL proteins were involved in outer membrane and LPS synthesis. Therefore, direct contact between wounds and LPS may lead to apoptosis, reduction in local cell proliferation, and delayed wound healing. Full article

Reproductive tract infections account for approximately 15% of male infertility cases. Escherichia coli (E. coli) represents the most frequently isolated bacterial strain in the semen of infertile men. All Gram-negative bacteria constitutively produce outer membrane vesicles (OMVs). The present study proved, for the first time, the involvement of OMVs in human sperm function. E. coli OMVs were isolated by ultracentrifugation and characterized via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Human sperm was exposed to OMVs (8 µg/mL) for different times (30, 45, 60 and 90 min). The vitality, motility, morphology, ROS level and DNA fragmentation of spermatozoa were evaluated. OMVs reduced the progressive motility and increased the immobile spermatozoa amount after 30 min of treatment. In addition, a significant increase in the percentage of intracellular ROS and sperm DNA fragmentation was recorded for each vesicular exposure time. These preliminary findings prove that OMVs contribute to altering human sperm function via two mechanisms: (i) impaired motility and (ii) DNA fragmentation. Full article