Effect Of Outer Membrane Vesicles Research Articles

Effect of outer membrane vesicles of Lactobacillus pentosus on Tau phosphorylation and CDK5-Calpain pathway in mice

Alzheimer's disease (AD) stands as a neurodegenerative disorder causing cognitive decline, posing a significant health concern for the elderly population in China. This study explored the effects of outer membrane vesicles (OMVs) from the gut microbiota of AD patients on learning and memory abilities and Tau protein phosphorylation in mice. In contrast to the OMVs from healthy controls and the PBS treatment group, mice treated with AD-OMVs exhibited notable declines in learning and memory capabilities, as evidenced by results from the Morris water maze, Y-maze, and novel object recognition tests. Immunohistochemistry and Western blot assessments unveiled elevated levels of hyperphosphorylated Tau in the cortex and hippocampus of mice treated with AD-OMVs. However, there were no alterations observed in the total Tau levels. In addition, AD-OMVs treated mice showed increased neuroinflammation indicated by elevated astrocytes and microglia. Molecular mechanism studies demonstrated that AD-OMVs could activate GSK3β, CDK5-Calpain and NF-κB pathways in mice hippocampus. These studies suggest AD patient gut microbiota derived OMVs can promote host Tau phosphorylation and improved neuroinflammation.

Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host’s immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-10, and TGF-β1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development.Key points• Preparation and characterization of P. multocida OMVs.• P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system.• P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Differential proteomic analysis and pathogenic effects of outer membrane vesicles derived from Acinetobacter baumannii under normoxia and hypoxia.

Acinetobacter baumannii is a major causative agent of nosocomial infections and its outer membrane vesicles (AbOMVs) have been shown to be involved in pathogenicity by transporting virulence factors and transferring information for communication between pathogens and host cells. Despite the fact that the infected sites of A. baumannii such as lungs and skin soft tissues are hypoxic, most studies on AbOMV virulence have used AbOMVs prepared under aerobic conditions. The present study aims to elucidate the protein profile and pathogenic impact of AbOMVs released under hypoxic condition. AbOMVs were isolated from A. baumannii under normoxic and hypoxic conditions, and their protein profiles were compared. The different effects of both normoxic and hypoxic AbOMVs in cytokine response from mouse macrophages, cytotoxicity to the human lung epithelial cells, and bacterial invasion were then investigated. Our results showed that A. baumannii under hypoxia released larger amounts of OMVs with different protein profiles. Although the cytotoxic effect of AbOMVs from normoxia and hypoxia were comparable, AbOMVs from normoxia induced higher TNF-α production and invasion of Staphylococcus aureus and Pseudomonas aeruginosa than those from hypoxia. On the other hand, AbOMVs significantly enhanced A. baumannii invasion into lung epithelial cells in a dose-dependent manner. These results clearly demonstrate that AbOMVs released from normoxic and hypoxic have different impacts in pathogenesis. This finding provides new insight into the complex interactions between A. baumannii, coinfecting pathogens and host cells via OMVs, in particular the different pathogenic effects of AbOMVs under normoxic and hypoxic conditions.

Outer membrane vesicles derived from heatstroke-associated intestinal microbiota promote multiple organ injury in mice.

Multiple organ injury is a common issue in heatstroke (HS); however, the underlying pathogenesis remains unclear. As an early event in HS, intestinal injury is an active participant that drives organ injury. Outer membrane vesicles (OMVs), a group of vesicles shed by unbalanced intestinal microbiota as "danger signals," mediate different functional cargo transport in cells and modulate varying biological events in distant target cells. However, the role of OMVs in HS-mediated organ damage remains unclear. Therefore, this study examined OMV production in HS and explored the effect of regulating multiple organ injury. To construct a mouse model, animals were exposed to hyperthermia. OMVs from the intestinal microbiota of HS and control mice were extracted by standardized differential ultracentrifugation. Thereafter, OMVs were characterized and infused into recipient mice via the tail vein. Cl-amidine (a pan-peptidylarginine deiminase inhibitor and OMV production inhibitor) was injected intraperitoneally (2mg/kg) 2h before HS treatment, and the absorption of HS OMVs by different organs was tracked. The effect of OMVs on inducing organ pathological changes, inflammatory infiltration, inflammatory cytokine expression, and serum organ injury biomarkers was demonstrated. HS increased OMV production by intestinal microbiota; OMVs were absorbed by different organs in vivo, and were especially enriched in the liver and lung. Compared to control OMVs, infusion with HS OMVs induced significant organ pathological changes, elevated inflammatory cell (macrophages and neutrophil) infiltration, inflammatory cytokine (TNF-ɑ, IL-1β, IL-6) expression, as well as serum biomarkers of organ injury. Similarly, inhibition of endogenous OMVs alleviated these organ injury indicators induced by HS. To our knowledge, the present study is the first to illustrate that OMVs induce acute organ impairment during severe HS, offering a foundation for subsequent studies and providing novel therapeutic targets.

An Investigation into the Effects of Outer Membrane Vesicles and Lipopolysaccharide of Porphyromonas gingivalis on Blood-Brain Barrier Integrity, Permeability, and Disruption of Scaffolding Proteins in a Human in vitro Model.

The effects of the key pathogens and virulence factors associated with gum disease such as Porphyromonas gingivalis (P.gingivalis) on the central nervous system is of great interest with respect to development of neuropathologies and hence therapeutics and preventative strategies. Chronic infections and associated inflammation are known to weaken the first line of defense for the brain, the blood-brain barrier (BBB). The focus of this study is to utilize an established human in vitro BBB model to evaluate the effects of P.gingivalis virulence factors lipopolysaccharide (LPS) and outer membrane vesicles (OMVs) on a primary-derived human model representing the neurovascular unit of the BBB. Changes to the integrity of the BBB after application of P.gingivalis LPS and OMVs were investigated and correlated with transport of LPS. Additionally, the effect of P.gingivalis LPS and OMVs on human brain microvascular endothelial cells in monolayer was evaluated using immunofluorescence microscopy. The integrity of the BBB model was weakened by application of P.gingivalis LPS and OMVs, as measured by a decrease in electrical resistance and a recovery deficit was seen in comparison to the controls. Application of P.gingivalis OMVs to a monoculture of human brain microvascular endothelial cells showed disruption of the tight junction zona occludens protein (ZO-1) compared to controls. These findings show that the integrity of tight junctions of the human BBB could be weakened by association with P.gingivalis virulence factors LPS and OMVs containing proteolytic enzymes (gingipains).

Immune Modulatory Effect of Outer Membrane Vesicles Derived from Salmonella on Mouse Bone Marrow-Derived Dendritic Cells

To study the effect of outer membrane vesicles (OMVs) derived from Salmonella typhimurium (ST) on the ultrastructural features and immune function of dendritic cells (DC). Mice bone marrow cells were collected aseptically, and myeloid DC were generated by the combined induction and amplification with recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Cell morphology was observed under inverted phase contrast microscope and the phenotype was identified with flow cytometry. ST-OMVs were isolated through ultracentrifugation. The survival rate of DC was assessed with CCK-8 assay, and the stimulus concentration of OMVs was henceforth determined. The ultrastructural characteristics of DC loaded with OMVs were observed with transmission electron microscopy. The cytokine secretion, surface molecule expression and phagocytic capacity of DC were examined with flow cytometry. The DC induced and amplified in vitro displayed typical DC phenotype in morphological analysis and the purity of DC exceeded 85%. Transmission electron microscopy showed that there were large numbers of protrusions on the cell surface. After stimulation with ST-OMVs, it was observed that the dendritic structures on the surface of DC were reduced and a large number of phagolysosomes were found in the cytoplasm. In addition, increased numbers of mitochondria, swelling and typical apoptosis were observed. After treatment with ST-OMVs at 5 μg/mL and 10 μg/mL, the secretion of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) of DC increased significantly ( P<0.05). Furthermore, the immature DC could differentiate into mature DCs after stimulation with ST-OMVs, which were characterized by a decrease in phagocytic capacity ( P<0.05) and an upregulation of phenotypic markers ( P<0.05). ST-OMVs can stimulate DC to produce TNF-α and IL-1β and promote DC maturation and antigen presentation.

The Attenuated Protective Effect of Outer Membrane Vesicles Produced by a mcr-1 Positive Strain on Colistin Sensitive Escherichia coli.

Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the “pre-antibiotic era”. Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.

Influence of outer membrane vesicles of Proteus mirabilis isolated from boar semen on sperm function

This study focuses on the effect of outer membrane vesicles (OMVs) in gram-negative bacteria on boar sperm function during in vitro storage. In the 40 ejaculates collected from Guangzhong Black boar, six gram-negative bacterial species were detected by 16S rDNA sequencing, of which Proteus mirabilis was the main contaminating bacterium. The OMVs of P. mirabilis were isolated by gradient ultracentrifugation. To reveal the effect of OMVs on boar sperm, different OMV concentrations were added to the Modena medium during sperm storage at 17 °C. Even after 3 days of storage, it was noted that low OMV dose (<5 μg/mL) in the extender did not significantly reduce sperm quality as compared with that in the control semen samples; however, sperm motility and sperm morphology were significantly altered in the extender owing to a high OMV dose (>10 μg/mL). The relative ROS level successively increased with OMV dose in sperm samples and storage time. Meanwhile, OMVs dramatically elevated the mitochondrial potential of sperm. OMVs could bind with the sperm membrane to further influence the capacity of sperm–oocyte binding; they also increased the expression of LC3 and caspase 3 and decreased that of anti-apoptosis-related protein, Bcl2, in sperm. It was concluded that OMVs of P. mirabilis influenced the function of boar sperm by inducing sperm membrane reconstruction as well as autophagy and apoptosis of sperm.

Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing.

Pathogens causing pneumonia utilize the complement regulator vitronectin to evade complement-mediated killing. Although vitronectin is associated with several chronic lung diseases, the role of bronchoalveolar vitronectin in pneumonia has not been studied. This study sought to reveal the involvement of vitronectin in the bronchoalveolar space during pneumonia, to assess the effect of outer membrane vesicles and endotoxin on vitronectin release, and to determine whether bacterial pathogens utilize pulmonary vitronectin for evasion. Vitronectin was analyzed in cell-free bronchoalveolar lavage fluid harvested from patients with pneumonia (n = 8) and from healthy volunteers after subsegmental endotoxin instillation (n = 13). Vitronectin binding by Pseudomonas aeruginosa and Haemophilus influenzae was analyzed, and subsequent complement evasion was assessed by serum challenge. The effects of outer membrane vesicles on vitronectin production in mouse lungs and human type II alveolar epithelial cells (A549) were determined. We detected increased vitronectin concentrations in lavage fluid during pneumonia (p = 0.0063) and after bronchial endotoxin challenge (p = 0.016). The capture of vitronectin by bacteria significantly reduced complement-mediated lysis. Following challenge with vesicles, vitronectin was detected in mouse bronchoalveolar space, and mouse alveolar epithelial cells in vivo as well as A549 cells in vitro contained increased levels of vitronectin. Taken together, outer membrane vesicles and endotoxin from Gram-negative bacteria induce vitronectin, which is released into the bronchoalveolar space, and used for evasion of complement-mediated clearance.

Vibrio cholerae Outer Membrane Vesicles Inhibit Bacteriophage Infection.

Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (phage) treatment has been proposed as an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown. Here, we show that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent. Our results suggest that phage therapy or prophylaxis should take into consideration the production of OMVs as a bacterial decoy mechanism that could influence the outcome of phage treatment.IMPORTANCE Phages have been increasingly recognized for the significance of their interactions with bacterial cells in multiple environments. Bacteria use myriad strategies to defend against phage infection, including restriction modification, abortive infection, phase variation of cell surface receptors, phage-inducible chromosomal islands, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems. The data presented here suggest that the apparently passive process of OMV release can also contribute to phage defense. By considering the effect of OMVs on V. cholerae infection by three unique virulent phages, ICP1, ICP2, and ICP3, we show that, in vitro, a reproducible reduction in bacterial killing is both dose and phage receptor dependent. This work supports a role for OMVs as natural decoys to defend bacteria from phage predation.

Effect of outer membrane vesicles derived from Escherichia coli on proliferation, apoptosis and migration of human neuroblastoma SK-N-SH cells in vitro

To investigate the effect of outer membrane vesicles derived from Escherichia coli on proliferation, apoptosis and migration of human neuroblastoma SK-N-SH cells in vitro. The outer membrane vesicles (OMVs) were obtained from wild-type Escherichia coli with ultracentrifugation method, and the morphology of the OMVs was observed by transmission electron microscopy and the vesicle diameter was determined using MALVERN ZEN3690. Human neuroblastoma SK-N-SH cells were treated with the OMVs at low (100 µg/mL), moderate (500 µg/mL) and high (1000 µg/mL) doses, and 24, 48 and 72 h later, the cell proliferation activity was detected with MTT assay. The expressions of apoptosis-related marker caspase-3 was detected using Western blotting, and TUNEL assay was performed to detect the cell apoptosis. The migration capacity of SK-N-SH cells was evaluated using Transwell migration assay. The isolated OMVs showed a circular or elliptical hollow structure with double-layer membrane and a diameter range of 30-450 nm. Compared with the control cells, SK-N-SH cells treated with the OMVs showed significantly lowered cell proliferation capacity with enhanced expression of caspase-3. Treatment of the cells with the OMVs resulted in increased cell apoptosis and significantly lowered migration capacity (P<0.05). The OMVs derived from Escherichia coli can produce cytotoxicity against SK-N-SH cells and might serve as a therapeutic agent for refractory neuroblastoma.

Outer Membrane Vesicles From Porphyromonas gingivalis Affect the Growth and Function of Cultured Human Gingival Fibroblasts and Umbilical Vein Endothelial Cells

The purpose of this study was to examine the effects of outer membrane vesicles (OMV) obtained from Porphyromonas gingivalis (Pg) on the growth and function of human gingival fibroblasts (HGF) and human umbilical vein endothelial cells (HUVEC). OMV were obtained from a cell-free growth medium of Pg ATCC 33277 by 40% NH2SO4 precipitation and ultracentrifugation. Cell proliferation was measured by 3H-thymidine incorporation into growing HGF and HUVEC. Endothelial cell function was determined by their capacity to form a network of capillary tubes on an extracellular matrix (ECM). Proliferating HGF and HUVEC demonstrated a significant dose-dependent inhibition of 3H-thymidine uptake when cultured with 0 to 40 microg/ml of OMV protein. HGF and HUVEC showed an IC50 of growth of about 9.0 microg/ml and 4.5 microg/ml of OMV protein, respectively. Capillary tube formation by HUVEC cultured on an ECM was suppressed by 70% to 80% with 5 microg/ml OMV protein after 18 hours of incubation. The presence of proteolytic enzymes in the OMV did not contribute to capillary tube disruption, since blocking enzyme activity with specific inhibitors did not reduce the suppression of capillary tube formation. After heating at 90 degrees C for 5 minutes, OMV significantly lost their capacity to suppress capillary tube formation. OMV significantly inhibit the proliferation of cultured HGF and HUVEC in a dose-dependent manner. OMV suppressed the capillary tube formation by cultured HUVEC. The factor(s) appeared to be a protein and not endotoxin because its inhibitory activity was markedly reduced by heat inactivation. These studies suggest that OMV contribute to chronic periodontitis by suppressing cell proliferation and revascularization in periodontal tissues.