Search Results (151)

Outer-membrane vesicles (OMVs), naturally secreted by Gram-negative bacteria, have gained recognition as a versatile platform for the development of next-generation vaccines. OMVs are essential contributors to bacterial pathogenesis, horizontal gene transfer, cellular communication, the maintenance of bacterial fitness, and quorum sensing. Their intrinsic immunogenicity, adjuvant properties, and scalability establish OMVs as potent tools for combating infectious diseases and cancer. Recent advancements in genetic engineering and biotechnology have further expanded the utility of OMVs, enabling the incorporation of multiple epitopes and antigens from diverse pathogens. These developments address critical challenges such as antigenic variability and co-infections, offering broader immune coverage and cost-effective solutions. This review explores the unique structural and immunological properties of OMVs, emphasizing their capacity to elicit robust immune responses. It critically examines established and emerging engineering strategies, including the genetic engineering of surface-displayed antigens, surface conjugation, glycoengineering, nanoparticle-based OMV engineering, hybrid OMVs, and in situ OMV production, among others. Furthermore, recent advancements in preclinical research on OMV-based vaccines, including synthetic OMVs, OMV-based nanorobots, and nanodiscs, as well as emerging isolation and purification methods, are discussed. Lastly, future directions are proposed, highlighting the potential integration of synthetic biology techniques to accelerate research on OMV engineering. 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

Extracellular vesicles (EVs) are nanometer-sized lipid structures actively secreted by Gram-negative and Gram-positive bacteria, representing a sophisticated microbial adaptation and communication strategy. These structures are involved in biomolecular transport, the regulation of biological processes, the modulation of host–pathogen interactions, and adaptation to hostile environmental conditions. EVs also play a crucial role in virulence, antibiotic resistance, and biofilm formation. This review will explore the biogenesis, composition, and biological mechanisms of outer membrane vesicles (OMVs) secreted by Gram-negative bacteria and membrane vesicles (MVs) generated by Gram-positive bacteria. In detail, the modulation of EVs in response to antibiotic exposure will be addressed. The role of EV morpho-functional adaptations will be studied in antimicrobial resistance, the gene determinant spread, and survival in adverse environments. This study aims to provide a comprehensive overview of the EV role in bacterial physiology, highlighting their ecological, evolutionary, and biotechnological implications. An overview of the enzymes and proteins mainly involved in OMV-mediated resistance mechanisms will also be provided. These insights could open new perspectives for developing therapeutic strategies that counteract EV secretion and biotechnological applications, such as vaccines and drug delivery systems. Full article

Background: Outer Membrane Vesicles (OMVs), nanosized particles derived from Gram-negative bacteria, are promising vaccine carriers due to innate immunogenicity and self-adjuvant properties. Yet the systematic evaluations of OMV-associated toxicity remain limited. Methods: We developed a CRISPR/Cas9-engineered Salmonella enterica serovar Typhimurium ΔmsbB mutant (Mut4_STM) to produce detoxified OMVs (Mut4_OMVs) with enhanced yield. Subcutaneous immunization of BALB/c mice with Mut4_OMVs to evaluate safety, and the adjuvant efficacy was also determined by injecting Mut4_OMVs with Yersinia pestis F1Vmut or Bacillus anthracis PA_D4 antigens, mixing formulation, respectively. Results: Mut4_OMVs showed a 9-fold protein yield increase over wild-type OMVs. While all mice injected with wild-type OMVs died, 100% survival was observed in those receiving Mut4_OMVs. However, dose-dependent pathological alterations became evident in specific organs as the administration dose escalated, such as induced splenic extramedullary hematopoiesis and renal edema. Despite residual toxicity, 2–3 doses of 10 μg Mut4_OMVs elicited antigen-specific antibody titers comparable to aluminum adjuvant controls and superior T-cell immune responses. Conclusion: While Mut4_OMVs retain potent adjuvant activity, their residual toxicity necessitates further biocompatibility optimization for safe vaccine applications. Full article

Bacterial outer membrane vesicles (OMVs) are nanoscale extracellular structures produced by Gram-negative bacteria that are critical for microbial biology and host-pathogen interactions and have great potential in biotechnological applications. Despite the availability of fluorescent dyes for OMV studies, many are repurposed from eukaryotic extracellular vesicle research and are not explicitly optimized for OMVs, leading to challenges in achieving consistent labeling, minimizing background noise, and preserving vesicle integrity during analyses. This study evaluates B2, a benzimidazole-derived fluorophore, for OMV labeling in advanced techniques like flow cytometry and confocal microscopy. OMVs were isolated from Escherichia coli strains BL21 and O157, and their integrity was confirmed using transmission electron microscopy (TEM). B2 staining protocols were optimized for OMVs, and fluorescence analyses revealed specific interactions with the vesicle membranes, reducing aggregation and enhancing signal uniformity. Flow cytometry indicated near-complete labeling efficiency (98–100%) with minimal background interference. Confocal microscopy further validated B2’s effectiveness, showing evident OMV internalization into epithelial HT-29 cells and compatibility with other fluorophores. Density functional theory (DFT) calculations, including Fukui function analysis, identified key electrophilic and nucleophilic regions in B2 that facilitate specific hydrogen bonding and polar interactions with membrane components. Non-covalent interaction (NCI) analysis revealed pronounced intramolecular hydrogen bonding along with discrete regions of weak van der Waals interactions. Molecular dynamics simulations suggest that B2 exhibits an affinity for both the hydrophobic core of the lipid bilayer and the core oligosaccharide region of the LPS layer, which collectively ensures sustained retention of the dye. The findings presented in this study position B2 as a valuable fluorophore for OMV research. Full article

Outer membrane vesicles (OMVs) are extracellular vesicles secreted by Gram-negative bacteria with diameters of 20–250 nm. OMVs contain various biologically active substances from their parent bacteria, such as proteins, lipids, and nucleic acids. Escherichia coli Nissle 1917 (EcN) is a Gram-negative probiotic that resides in the human intestine. EcN-derived OMVs are pivotal in modulating intestinal immune responses. However, few studies have addressed the heterogeneity of EcN-derived OMVs in terms of size, significantly limiting the research on their clinical applications. Currently, there are a lack of feasible methods for obtaining EcN-derived OMVs of different sizes. To address this knowledge gap, we developed a membrane filtration method to isolate EcN-derived OMVs of varying sizes. In this study, we first used gradient filtration to isolate high-purity EcN-derived OMVs and conducted a proteomic analysis. Subsequently, we used membrane filtration to separate the EcN-derived OMVs by size. We successfully obtained EcN-derived OMVs of three specific sizes: <50 nm, 50–100 nm, and 100–300 nm. We then performed proteomic analyses of these EcN-derived OMVs and compared their protein profiles. Finally, we compared the ability of each EcN-derived OMV type to induce RAW264.7 macrophages to secrete the pro-inflammatory factor interleukin (IL)-1β and the anti-inflammatory factor IL-10. The EcN-derived OMVs contained 646 different proteins overall; those of different sizes contained different protein types. Among them, the EcN-derived OMVs in the <50 nm group contained significantly fewer proteins (262 different types in total) than those in the 50–100 nm (1603 types) and 100–300 nm (1568 types) groups. Furthermore, the <50 nm group had fewer membrane proteins (40) than the 50–100 nm (215) and 100–300 nm (209) groups. We also found that RAW264.7 macrophages secreted different concentrations of IL-1β and IL-10 following co-incubation with the three EcN-derived OMV types. The 50–100 nm EcN-derived OMV group showed a stronger effect in terms of inducing inflammatory cytokine secretion compared to the other two groups. This study provides direct experimental evidence that EcN-derived OMVs of different sizes exhibit heterogeneous properties. Full article

Salmonella enterica serovar Typhi (S. Typhi) produces outer membrane vesicles (OMVs) that remain comparatively underexplored as potential biotechnological tools. Here, we investigated how hypervesiculating S. Typhi mutants (ΔtolR and ΔdegS) can be engineered to load and deliver the fluorescent reporter protein mCherry, targeting human epithelial cells and the murine immune system. Deletions in tolR and degS led to distinct OMV phenotypes characterized by higher vesicle production and altered cargo composition, underscoring the impact of disrupted membrane integrity and envelope stress on OMV biogenesis. By fusing mCherry with the S. Typhi OmpA signal peptide (SP_ompA), we achieved robust and functionally intact intravesicular packaging in all strains. Flow cytometry and confocal microscopy revealed that the ΔtolR mutant exhibited particularly high cargo loading in the OMV fraction and pronounced mCherry delivery to epithelial cells, highlighting the potential of hypervesiculation to enhance OMV-based protein transport. However, immunization studies in mice showed that wild-type OMVs, despite carrying less mCherry than their hypervesiculating counterparts, induced the strongest anti-mCherry IgG responses. These findings indicate that, at least under these conditions, antigen loading alone is not sufficient to fully determine immunogenicity. Instead, the intrinsic composition or adjuvant-like properties of OMVs play a pivotal role in driving robust immune activation. Our results establish S. Typhi OMVs, especially when genetically modified with a Sec-dependent targeting signal (SP_ompA), as versatile platforms for heterologous protein delivery. Although hypervesiculation facilitates increased protein encapsulation and delivery to epithelial cells, native OMVs appear to better preserve and/or present antigens for effective immunogenic responses in vivo. These insights set the stage for further optimization of S. Typhi OMVs in vaccine development and protein therapeutics, where balancing cargo loading with immunostimulatory features may be key to achieving maximal efficacy. Full article

Gram-negative bacteria release outer membrane vesicles (OMVs) that deliver various molecules, including virulence factors, to interact with their host. Recent studies have suggested that OMVs may also serve as carriers for RNAs, particularly small regulatory noncoding RNAs (sRNAs). For these RNAs to function effectively, they typically require a protein cofactor, Hfq, known as an RNA chaperone. In previous work, using molecular imaging, Circular Dichroism CD, and InfraRed FTIR spectroscopies, we demonstrated that Hfq interacts with the bacterial inner membrane and forms pores, suggesting a possible role in translocating RNA from the cytoplasm to periplasm and then to OMVs. In this study, we expand on our previous findings and provide evidence that RNA molecules bind to the Escherichia coli inner membrane in an Hfq-dependent manner. Moreover, we show that the lipid nature, in particular the presence of a cardiolipin-rich domain, is crucial for this interaction. These results reveal a new aspect of RNA translocation through the inner membrane, for further packaging in OMVs, and underscore the importance of Hfq in this mechanism. Full article

Increased abundance of Segatella copri (S. copri) within the gut microbiota is associated with systemic inflammatory diseases, including rheumatoid arthritis. Although outer-membrane vesicles (OMVs) of Gram-negative bacteria are important players in microbiota–host communication, the effect of S. copri-derived OMVs on immune cells is unknown. Macrophages engulf and eliminate foreign material and are conditioned by environmental signals to promote either homeostasis or inflammation. Thus, we aimed to explore the impact of S. copri-OMVs on human macrophages in vitro, employing THP-1 and monocyte-derived macrophage models. The uptake of DiO-labeled S. copri-OMVs into macrophages was monitored by confocal microscopy and flow cytometry. Furthermore, the effect of S. copri and S. copri-OMVs on the phenotype and cytokine secretion of naïve (M0), pro-inflammatory (M1), and anti-inflammatory (M2) macrophages was analyzed by flow cytometry and ELISA, respectively. We show that S. copri-OMVs enter human macrophages through macropinocytosis and clathrin-dependent mechanisms. S. copri-OMVs, but not the parental bacterium, induced a dose-dependent increase in the expression of M1-related surface markers in M0 and M2 macrophages and activated the secretion of large amounts of pro-inflammatory cytokines in M1 macrophages. These results highlight an important role of S. copri-OMVs in promoting pro-inflammatory macrophage responses, which might contribute to systemic inflammatory diseases. Full article

Introduction: Osteoporosis (OP) is a prevalent condition marked by reduced bone density and a heightened risk of fractures. Current treatments often have side effects, underscoring the need for safer alternatives. Recent research highlights the significant role of gut microbiota and their metabolites in maintaining bone health. Notably, bacterial outer membrane vesicles (OMVs) have emerged as a promising platform due to their nanoscale sizes, low toxicity, drug-loading capabilities, and excellent biocompatibility. Methods: In this study, we developed a delivery system using OMVs derived from Pseudomonas mirabilis (PM). By anchoring bone-targeting peptides to the PM-OMVs membrane, we equipped these vesicles to deliver endogenous miRNAs to the bone microenvironment effectively. Results and Discussion: The bone-targeted PM-OMVs (PM-OMVs-BT) demonstrated exceptional bone-targeting abilities and exhibited a favorable safety profile in vivo. Additionally, LGG-OMVs-BT were successfully internalized by bone marrow stromal cells (BMSCs) without significant cytotoxicity, effectively promoting their osteogenic differentiation and mineralization. In conclusion, our study indicates that PM-OMVs-BT could offer a safe and effective treatment option for OP. Full article

Infection with Porphyromonas gingivalis (Pg), which is a major periodontal pathogen, causes a large number of systemic diseases based on chronic inflammation such as diabetes and Alzheimer’s disease (AD). However, it is not yet fully understood how Pg can augment local systemic immune and inflammatory responses during progression of AD. There is a strong association between depression and elevated levels of inflammation. Noradrenaline (NA) is a key neurotransmitter that modulates microglial activation during stress conditions. In this study, we have thus investigated the regulatory mechanisms of NA on the production of interleukin-1β (IL-1β) by microglia following stimulation with Pg virulence factors, lipopolysaccharide (LPS), and outer membrane vesicles (OMVs). NA (30–1000 nM) significantly enhanced the mRNA level, promoter activity, and protein level of IL-1β up to 20-fold in BV-2 microglia following treatment with Pg LPS (10 μg/mL) and OMVs (150 μg of protein/mL) in a dose-dependent manner. Pharmacological studies have suggested that NA synergistically augments the responses induced by Pg LPS and OMVs through different mechanisms. AP-1 is activated by the β₂ adrenergic receptor (Aβ₂R)-mediated pathway. NF-κB, which is activated by the Pg LPS/toll-like receptor 2-mediated pathway, is required for the synergistic effect of NA on the Pg LPS-induced IL-1β production by BV-2 microglia. Co-immunoprecipitation combined with Western blotting and the structural models generated by AlphaFold2 suggested that cross-coupling of NF-κB p65 and AP-1 c-Fos transcription factors enhances the binding of NF-κB p65 to the IκB site, resulting in the synergistic augmentation of the IL-1β promoter activity. In contrast, OMVs were phagocytosed by BV-2 microglia and then activated the TLR9/p52/RelB-mediated pathway. The Aβ₂R/Epac-mediated pathway, which promotes phagosome maturation, may be responsible for the synergistic effect of NA on the OMV-induced production of IL-1β in BV-2 microglia. Our study provides the first evidence that NA synergistically enhances the production of IL-1β in response to Pg LPS and OMVs through distinct mechanisms. Full article

Pseudoalteromonas species are recognized for their probiotic roles in reducing pathogens in aquaculture products by secreting a broad range of antimicrobial compounds. Some species, like P. piscicida, are also predators that attack susceptible prey bacteria, including V. parahaemolyticus, by transferring outer membrane vesicles (OMVs) containing digestive compounds to the surface of their prey. These vesicles digest holes in the prey’s cell wall releasing nutrients upon which the Pseudoalteromonas feed. In the present study, scanning electron microscopy was performed on two P. piscicida strains grown in sterile seawater and nutrient-enriched seawater, without the presence of bacterial prey, to determine if the presence of prey or low-nutrient media was required to induce vesicle formation. Micrographs revealed OMV formation and high pleomorphism of P. piscicida in the absence of prey cells and regardless of the nutrient levels of the seawater. Phenotypic characteristics included the presence of (i) vesiculated and non-vesiculated bacteria, (ii) large bulbous OMV versus small OMV, (iii) pilus-like connectors of widely varying lengths to which vesicles were attached, (iv) highly elongated (10 µm long) Pseudoalteromonas cells, and (v) cells that appeared to extend to 50 µm long and to be septating and dividing into short chains and individual cells. The possible contribution of these novel phenotypes to Pseudoalteromonas predation is discussed. Full article

Outer membrane vesicles (OMVs) are double-layered structures of nanoscale lipids released by gram-negative bacteria. They have the same membrane composition and characteristics as primitive cells, which enables them to penetrate cells and tissues efficiently. These OMVs exhibit excellent membrane stability, immunogenicity, safety, and permeability (which makes it easier for them to penetrate into tumour tissue), making them suitable for developing cancer vaccines and drug delivery systems. Recent studies have focused on engineering OMVs to enhance tumour-targeting capabilities, reduce toxicity, and extend circulation time in vivo. This article reviews the latest progress in OMV engineering for tumour treatment and discusses the challenges associated with the use of OMV-based antitumour therapy in clinical practice. Full article

The high sensitivity of living organic forms to space radiation remains the critical issue during spaceflight, to which they will be chronically exposed during months of interplanetary or even decades of interstellar spaceflight. In the human body, all actively dividing and poorly differentiated cells are always close to being damaged by radiological or chemical agents. The chronic exposure to ionizing radiation primarily causes changes in blood counts and intestinal damage such as fibrosis, obliterative vasculitis, changes in the gut microbiota, and atrophy or degeneration of muscle fibers. The project “MISS: Microbiome Induced Space Suit” was presented at the Giant Jamboree of the International Genetically Engineered Machine Competition 2021, with the aim to investigate the ability of the novel microbiota-mediated approach to enhance human resistance to ionizing radiation. The key innovative part of the project was the idea to create a novel radioprotector delivery mechanism based on human gut microbiota with the function of outer membrane vesicles (OMVs) secretion. The project concept proposed the feasibility of genetically modifying the human microbiota in situ through the delivery of genetic constructs to the host’s crypts using silicon nanoparticles with chemically modified surfaces. In this perspective, we discuss the advances in modifying microbiota-mediated secretory activity as a promising approach for radioprotection and as an alternative to hormone therapy and other health conditions that currently require continuous drug administration. Future clinical trials of in situ methods to genetic engineering the crypt microbiota may pave the way for indirect regulation of human cells. Full article

Oral squamous-cell carcinoma (OSCC) poses significant treatment challenges due to its high recurrence rates and the limitations of current therapies. Titanium dioxide (TiO₂) nanoparticles are promising radiosensitizers, while bacterial outer membrane vesicles (OMVs) are known for their immunomodulatory properties. This study investigates the potential of OMV-encapsulated TiO₂ nanoparticles (TiO₂@OMV) to combine these effects for improved OSCC treatment. TiO₂ nanoparticles were synthesized using a hydrothermal method and encapsulated within OMVs derived from Escherichia coli. The TiO₂@OMV carriers were evaluated for their ability to enhance radiosensitivity and stimulate immune responses in OSCC cell lines. Reactive oxygen species (ROS) production, macrophage recruitment, and selective cytotoxicity toward cancer cells were assessed. TiO₂@OMV demonstrated significant radiosensitization and immune activation compared to unencapsulated TiO₂ nanoparticles. The system selectively induced cytotoxicity in OSCC cells, sparing normal cells, and enhanced ROS generation and macrophage-mediated antitumor responses. This study highlights TiO₂@OMV as a dual-action therapeutic platform that synergizes radiotherapy and immunomodulation, offering a targeted and effective strategy for OSCC treatment. The approach could improve therapeutic outcomes and reduce the adverse effects associated with conventional therapies. Full article