Methods for the Characterization of the Colloidal Properties of Bacterial Membrane Vesicles.

Bacterial membrane vesicles (BMVs) are extracellular vesicles secreted by either Gram-positive or Gram-negative bacteria. These BMVs typically possess a diameter between 20 and 250 nm. Due to their size, when these BMVs are suspended in another medium, they could be constituents of a colloidal system. It has been hypothesized that investigating BMVs as colloidal particles could help characterize BMV interactions with other environmentally relevant surfaces. Developing a more thorough understanding of BMV interactions with other surfaces would be critical for developing predictive models of their environmental fate. However, this bio-colloidal perspective has been largely overlooked for BMVs, despite the wealth of methods and expertise available to characterize colloidal particles. A particular strength of taking a more colloid-centric approach to BMV characterization is the potential to quantify a particle's attachment efficiency (α). These values describe the likelihood of attachment during particle-particle or particle-surface interactions, especially those interactions which are governed by physicochemical interactions (such as those described by DLVO and xDLVO theory). Elucidating the influence of physical and electrochemical properties on these attachment efficiency values could give insights into the primary factors driving interactions between BMVs and other surfaces. This chapter details methods for the characterization of BMVs as colloids, beginning with size and surface charge (i.e., electrophoretic mobility/zeta potential) measurements. Afterward, this chapter will address experimental design, especially column experiments, targeted for BMV investigation and the determination of α values.

Metal ions-anchored bacterial outer membrane vesicles for enhanced ferroptosis induction and immune stimulation in targeted antitumor therapy.

The activation of ferroptosis presents a versatile strategy for enhancing the antitumor immune responses in cancer therapy. However, developing ferroptosis inducers that combine high biocompatibility and therapeutic efficiency remains challenging. In this study, we propose a novel approach using biological nanoparticles derived from outer membrane vesicles (OMVs) of Escherichia coli for tumor treatment, aiming to activate ferroptosis and stimulate the immune responses. Specifically, we functionalize the OMVs by anchoring them with ferrous ions via electrostatic interactions and loading them with the STING agonist-4, followed by tumor-targeting DSPE-PEG-FA decoration, henceforth referred to as OMV/SaFeFA. The anchoring of ferrous ions endows the OMVs with peroxidase-like activity, capable of inducing cellular lipid peroxidation by catalyzing H2O2 to •OH. Furthermore, OMV/SaFeFA exhibits pH-responsive release of ferrous ions and the agonist, along with tumor-targeting capabilities, enabling tumor-specific therapy while minimizing side effects. Notably, the concurrent activation of the STING pathway and ferroptosis elicits robust antitumor responses in colon tumor-bearing mouse models, leading to exceptional therapeutic efficacy and prolonged survival. Importantly, no acute toxicity was observed in mice receiving OMV/SaFeFA treatments, underscoring its potential for future tumor therapy and clinical translation.

TrxR1 is involved in the activation of Caspase-11 by regulating the oxidative-reductive status of Trx-1.

Sepsis is a common complication of infections that significantly impacts the survival of critically patients. Currently, effective pharmacological treatment strategies are lacking. Auranofin, known as an inhibitor of Thioredoxin reductase (TrxR), exhibits anti-inflammatory activity, but its role in sepsis is not well understood. Here, we demonstrate the significant inhibitory effect of Auranofin on sepsis in a cecal ligation and puncture (CLP) mouse model. In vitro, Auranofin inhibits pyroptosis triggered by Caspase-11 activation. Further investigations reveal that inhibiting TrxR1 suppresses macrophage pyroptosis induced by E. coli, while TrxR2 does not exhibit this effect. TrxR1, functioning as a reductase, regulates the oxidative-reductive status of Thioredoxin-1 (Trx-1). Mechanistically, the modulation of Trx-1's reductive activity by TrxR1 may be involved in Caspase-11 activation-induced pyroptosis. Additionally, inhibiting TrxR1 maintains Trx-1 in its oxidized state. The oxidized form of Trx-1 interacts with Caveolin-1 (CAV1), regulating outer membrane vesicle (OMV) internalization. In summary, our study suggests that inhibiting TrxR1 suppresses OMV internalization by maintaining the oxidized form of Trx-1, thereby restricting Caspase-11 activation and alleviating sepsis.

Bacterial outer membrane vesicle nanorobot.

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Budding and explosive membrane vesicle production by hypervesiculating Escherichia coli strain ΔrodZ.

Escherichia coli produces extracellular vesicles called outer membrane vesicles. In this study, we investigated the mechanism underlying the hypervesiculation of deletion mutant ΔrodZ of E. coli. RodZ forms supramolecular complexes with actin protein MreB and peptidoglycan (PG) synthase, and plays an important role in determining the cell shape. Because mreB is an essential gene, an expression-repressed strain (mreB R3) was constructed using CRISPRi, in which the expression of mreB decreased to 20% of that in the wild-type (WT) strain. In shaken-flask culture, the ΔrodZ strain produced >50 times more vesicles than the WT strain. The mreB-repressed strain mreB R3 showed eightfold higher vesicle production than the WT. ΔrodZ and mreB R3 cells were observed using quick-freeze replica electron microscopy. As reported in previous studies, ΔrodZ cells were spherical (WT cells are rod-shaped). Some ΔrodZ cells (around 7% in total) had aberrant surface structures, such as budding vesicles and dented surfaces, or curved patterns on the surface. Holes in the PG layer and an increased cell volume were observed for ΔrodZ and mreB R3 cells compared with the WT. In conditions of osmotic support using sucrose, the OD660 value of the ΔrodZ strain increased significantly, and vesicle production decreased drastically, compared with those in the absence of sucrose. This study first clarified that vesicle production by the E. coli ΔrodZ strain is promoted by surface budding and a burst of cells that became osmotically sensitive because of their incomplete PG structure.

Bacterial outer membrane vesicle-cancer cell hybrid membrane-coated nanoparticles for sonodynamic therapy in the treatment of breast cancer bone metastasis.

Breast cancer bone metastasis is a terminal-stage disease and is typically treated with radiotherapy and chemotherapy, which causes severe side effects and limited effectiveness. To improve this, Sonodynamic therapy may be a more safe and effective approach in the future. Bacterial outer membrane vesicles (OMV) have excellent immune-regulating properties, including modulating macrophage polarization, promoting DC cell maturation, and enhancing anti-tumor effects. Combining OMV with Sonodynamic therapy can result in synergetic anti-tumor effects. Therefore, we constructed multifunctional nanoparticles for treating breast cancer bone metastasis. We fused breast cancer cell membranes and bacterial outer membrane vesicles to form a hybrid membrane (HM) and then encapsulated IR780-loaded PLGA with HM to produce the nanoparticles, IR780@PLGA@HM, which had tumor targeting, immune regulating, and Sonodynamic abilities. Experiments showed that the IR780@PLGA@HM nanoparticles had good biocompatibility, effectively targeted to 4T1 tumors, promoted macrophage type I polarization and DC cells activation, strengthened anti-tumor inflammatory factors expression, and presented the ability to effectively kill tumors both in vitro and in vivo, which showed a promising therapeutic effect on breast cancer bone metastasis. Therefore, the nanoparticles we constructed provided a new strategy for effectively treating breast cancer bone metastasis.

Proteomic Analysis of Caco-2 Cells Disrupted by EcN 1917-Derived OMVs Reveals Molecular Information on Bacteria-Mediated Cancer Cell Migration.

Escherichia coli Nissle 1917 (EcN 1917) exhibits distinct tumor-targeting activity, and early studies demonstrated that outer membrane vesicles (OMVs) mediate bacteria-host interactions. To decipher the molecular mechanism underlying the interaction between EcN 1917 and host cells via OMV-mediated communication, we investigated the phenotypic changes in Caco-2 cells perturbed by EcN 1917-derived OMVs and constructed proteomic maps of the EcN 1917-derived OMV components and OMV-perturbed host cells. Our findings revealed that the size of the EcN 1917-derived OMV proteome increased 4-fold. Treatment with EcN 1917-derived OMVs altered the proteomic and phosphoproteomic profiles of host cells. Importantly, for the first time, we found that treatment with EcN 1917-derived OMVs inhibited cancer cell migration by suppressing the expression of ANXA9. In addition, phosphoproteomic data suggested that the ErbB pathway may be involved in OMV-mediated cell migration. Taken together, our study provides valuable data for further investigations of OMV-mediated bacteria-host interactions and offers great insights into the underlying mechanism of probiotic-assisted colorectal cancer therapy.

Lysosome-Targeting Bacterial Outer Membrane Vesicles for Tumor Specific Degradation of PD-L1.

Increased expression of immune check point genes, such as PD-L1, is one of the main reasons for immunosuppression, especially for colon cancer. Development of novel therapeutic strategies is of great importance to improve the prognosis. In this study, outer membrane vesicles (OMV) derived from Gram-negative bacteria are engineered to immune checkpoint blockade nanosystem for efficient elicitation of anti-tumor immunity. Briefly, the OMVs are engineered with Lyp1-Traptavidin (S52G, R53D mutant of streptavidin) fusion protein displayed on the surface. The Lyp-1 endows the OMV with the capacity to target tumor tissues, while the Traptavidin ensures easy decoration of biotinylated anti-PD-L1 and biotinylated M6P (mannose 6-phosphate). The simultaneously anchored anti-PD-L1 and M6P (ligand for cation-independent mannose 6-phosphate receptor) on the engineered OMVs coordinately direct the membrane PD-L1 to lysosome for degradation, and thus unleash the anti-tumor immunity. With syngeneic tumor model, the engineered OMVs are confirmed to boost immunity, inhibit cancer growth, and thus prolong survival. Together, A proposed OMV-based modular nanosystem that enables assembly of biotinylated anti-PD-L1 and M6P on the surface for tumor-targeted immune checkpoint blockade.

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.

Studies on the mechanisms of Helicobacter pylori inhibition by Syzygium aromaticum aqueous extract.

BACKGROUND: The aqueous extract of the dried buds of Syzygium aromaticum (SAAE) have the potential to alleviate Helicobacter pylori infection, but the specific molecular mechanism has not been fully elucidated. PURPOSE: This study aimed to investigate the underlying mechanisms of SAAE on H. pylori pathogenicity. METHODS: The inhibitory kinetics and anti-H. pylori adhesive capacity assays were conducted to examine the effects of SAAE on the growth and adhesive capability of H. pylori. The H. pylori outer membrane vesicles (OMVs) were purified from the culture supernatant through high-speed centrifugation, filtration, and two rounds of ultracentrifugation. Their characteristics and protein composition were then identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and qualitative proteomics study. Subsequently, the effect of SAAE on the pathogenicity of H. pylori OMVs was investigated using the Griess reagent assay, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics study, TEM, and western blotting assay. RESULTS: SAAE exhibited inhibitory effects on H. pylori growth and adhesion. The isolated H. pylori OMVs showed particle size of 27-242 nm and Zeta potential of -9.67 ± 0.53 mV. A total of 599 proteins were identified in the OMVs. Proteomics study indicated that the differential expressed proteins induced by OMVs with or without SAAE commonly enriched in P53 and autophagy pathways. Besides, SAAE counteracted the increased production of pro-inflammatory cytokines and attenuated the induction of cell autophagy caused by H. pylori OMVs. Furthermore, SAAE normalized the abnormal regulation of downstream targets (AIFM2 and IGFBP3) in the P53 signaling pathway caused by H. pylori OMVs. CONCLUSION: SAAE can inhibit the growth and adhesion of H. pylori, reduce the inflammation and autophagy induced by H. pylori OMVs, and combated the abnormal regulation of P53 signaling pathway caused by H. pylori OMVs. These findings may help elucidate the mechanisms through which SAAE reduces the pathogenicity of H. pylori.

Outer membrane vesicle-wrapped manganese nanoreactor for augmenting cancer metalloimmunotherapy through hypoxia attenuation and immune stimulation.

Tumor hypoxia, high oxidative stress, and low immunogenic create a deep-rooted immunosuppressive microenvironment, posing a major challenge to the therapeutic efficiency of cancer immunotherapy for solid tumor. Herein, an intelligent nanoplatform responsive to the tumor microenvironment (TME) capable of hypoxia relief and immune stimulation has been engineered for efficient solid tumor immunotherapy. The MnO2@OxA@OMV nanoreactor, enclosing bacterial-derived outer membrane vesicles (OMVs)-wrapped MnO2 nanoenzyme and the immunogenic cell death inducer oxaliplatin (OxA), demonstrated intrinsic catalase-like activity within the TME, which effectively catalyzed the endogenous H2O2 into O2 to enable a prolonged oxygen supply, thereby alleviating the tumor's oxidative stress and hypoxic TME, and expediting OxA release. The combinational action of OxA-caused ICD effect and Mn2+ from nanoreactor enabled the motivation of the cGAS-STING pathway to significantly improve the activation of STING and dendritic cells (DCs) maturation, resulting in metalloimmunotherapy. Furthermore, the immunostimulant OMVs played a crucial role in promoting the infiltration of activated CD8+T cells into the solid tumor. Overall, the nanoreactor offers a robust platform for solid tumor treatment, highlighting the significant potential of combining relief from tumor hypoxia and immune stimulation for metalloimmunotherapy. STATEMENT OF SIGNIFICANCE: A tailor-made nanoreactor was fabricated by enclosing bacterial-derived outer membrane vesicles (OMVs) onto MnO2 nanoenzyme and loading with immunogenic cell death inducer oxaliplatin (OxA) for tumor metalloimmunotherapy. The nanoreactor possesses intrinsic catalase-like activity within the tumor microenvironment, which effectively enabled a prolonged oxygen supply by catalyzing the conversion of endogenous H2O2 into O2, thereby alleviating tumor hypoxia and expediting OxA release. Furthermore, the TME-responsive release of nutritional Mn2+ sensitized the cGAS-STING pathway and collaborated with OxA-induced immunogenic cell death (ICD). Combing with immunostimulatory OMVs enhances the uptake of nanoreactors by DCs and promotes the infiltration of activated CD8+T cells. This nanoreactor offers a robust platform for solid tumor treatment, highlighting the significant potential of combining relief from tumor hypoxia and immune stimulation for metalloimmunotherapy.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Sub-MIC Antibiotics Modulate Productions of Outer Membrane Vesicles in Tigecycline-Resistant Escherichia coli.

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.

Outer membrane vesicles as a platform for the discovery of antibodies to bacterial pathogens.

Bacterial outer membrane vesicles (OMVs) are nanosized spheroidal particles shed by gram-negative bacteria that contain biomolecules derived from the periplasmic space, the bacterial outer membrane, and possibly other compartments. OMVs can be purified from bacterial culture supernatants, and by genetically manipulating the bacterial cells that produce them, they can be engineered to harbor cargoes and/or display molecules of interest on their surfaces including antigens that are immunogenic in mammals. Since OMV bilayer-embedded components presumably maintain their native structures, OMVs may represent highly useful tools for generating antibodies to bacterial outer membrane targets. OMVs have historically been utilized as vaccines or vaccine constituents. Antibodies that target bacterial surfaces are increasingly being explored as antimicrobial agents either in unmodified form or as targeting moieties for bactericidal compounds. Here, we review the properties of OMVs, their use as immunogens, and their ability to elicit antibody responses against bacterial antigens. We highlight antigens from bacterial pathogens that have been successfully targeted using antibodies derived from OMV-based immunization and describe opportunities and limitations for OMVs as a platform for antimicrobial antibody development. KEY POINTS: • Outer membrane vesicles (OMVs) of gram-negative bacteria bear cell-surface molecules • OMV immunization allows rapid antibody (Ab) isolation to bacterial membrane targets • Review and analysis of OMV-based immunogens for antimicrobial Ab development.

Safe Induction of Acute Inflammation with Enhanced Antitumor Immunity by Hydrogel-Mediated Outer Membrane Vesicle Delivery.

Acute inflammation has the potential for the recruitment of immune cells, inhibiting tumor angiogenesis, metastasis, and drug resistance thereby overcoming the tumor immunosuppressive microenvironment caused by chronic inflammation. Here, an acute inflammation inducer using bacteria outer membrane vesicles (OMVs) loaded in thermal-sensitive hydrogel (named OMVs-gel) for localized and controlled release of OMVs in tumor sites is proposed. OMVs trigger neutrophil recruitment and amplify acute inflammation inside tumor tissues. The hydrogel ensures drastic inflammation is confined within the tumor, addressing biosafety concerns that the direct administration of free OMVs may cause fatal effects. This strategy eradicated solid tumors safely and rapidly. The study further elucidates one of the possible immune mechanisms of OMVs-gel therapy, which involves the assembly of antitumor neutrophils and elastase release for selective tumor killing. Additionally, tumor vascular destruction induced by OMVs-gel results in tumor darkening, allowing for combinational photothermal therapy. The findings suggest that the use of OMVs-gel can safely induce acute inflammation and enhance antitumor immunity, representing a promising strategy to promote acute inflammation application in tumor immunotherapy.

Outer membrane vesicles of avian pathogenic Escherichia coli induce necroptosis and NF-κB activation in chicken macrophages via RIPK1 mediation.

Outer membrane vesicles (OMVs) are soluble mediators secreted by Gram-negative bacteria that are involved in communication. They can carry a variety of harmful molecules, which induce cytotoxic responses and inflammatory reactions in the absence of direct host cell-bacterium interactions. We previously reported the isolation of OMVs from avian pathogenic Escherichia coli (APEC) culture medium by ultracentrifugation, and characterized them as a substance capable of inducing the production of pro-inflammatory cytokines and causing tissue damage. However, the specific mechanisms by which APEC-secreted OMVs activate host cell death signaling and inflammation are poorly understood. Here, we show that OMVs are involved in the pathogenesis of APEC disease. In an APEC/chicken macrophage (HD11) coculture system, APEC significantly promoted HD11 cell death and inflammatory responses by secreting OMVs. Using western blotting analysis and specific pathway inhibitors, we demonstrated that the induction of HD11 death by APEC OMVs is associated with the activation of receptor interacting serine/threonine kinase 1 (RIPK1)-, receptor interacting serine/threonine kinase 3 (RIPK3)-, and mixed lineage kinase like pseudokinase (MLKL)-induced necroptosis. Notably, necroptosis inhibitor-1 (Nec-1), an RIPK1 inhibitor, reversed these effects. We also showed that APEC OMVs promote the activation of the NF-κB signaling pathway, leading to the phosphorylation of IκB-α and p65, the increased nuclear translocation of p65, and the significant upregulation of interleukin 1ß (IL-1ß) and IL-6 transcription. Importantly, APEC OMVs-induced IL-1ß and IL-6 mRNA expression and the activation of the NF-κB signaling pathway were similarly significantly inhibited by a RIPK1-specific inhibitor. Based on these findings, we have established that RIPK1 plays a dual role in HD11 cells necroptosis and the proinflammatory cytokine (IL-1ß and IL-6) expression induced by APEC OMVs. RIPK1 mediated the induction of necroptosis and the activation of the NF-κB in HD11 cells via APEC OMVs. The results of this study provide a basis for further investigation of the contribution of OMVs to the pathogenesis of APEC.

Proteomic analysis of meropenem-induced outer membrane vesicles released by carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important multidrug resistance (MDR) pathogen that threatens human health and is the main source of hospital-acquired infection. Outer membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria and contain materials involved in bacterial survival and pathogenesis. They also contribute to cellular communication to nearby or distant recipient cells and influence their functions and phenotypes. In this study, we sought to understand the mechanism of bacterial response to meropenem pressure and explore the relationship between pathogenic proteins and the high pathogenicity of bacteria. We performed whole-genome PacBio sequencing on a clinical CRKP strain, and its OMVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and proteomic analysis. Thousands of vesicle proteins have been identified in mass spectrometry-based high-throughput proteomics analyses of K. pneumoniae OMVs. Protein functionality analysis showed that the OMVs were predominantly involved in metabolic, intracellular compartments, nucleic acid binding, survival, defense, and antibiotic resistance, such as Chromosome partition protein MukB, 3-methyl-2-oxobutanoate hydroxymethyltransferase, methionine-tRNA ligase, Heat shock protein 60 family chaperone GroEL, and Gamma-glutamyl phosphate reductase. Additionally, a protein-protein interaction network demonstrated that OMVs from meropenem-treated K. pneumoniae showed the highest connectivity in DNA polymerase I, phenylalanine-tRNA ligase beta subunit, DNA-directed RNA polymerase subunit beta, methionine-tRNA ligase, DNA-directed RNA polymerase subunit beta, and DNA-directed RNA polymerase subunit alpha. The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-treated K. pneumoniae, which provides clues for revealing the biogenesis and pathophysiological functions of Gram-negative bacteria OMVs. The significant differentially expressed proteins identified in this study are of great significance for exploring effective control strategies for CRKP infection.IMPORTANCEMeropenem is one of the main antibiotics used in the clinical treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP). This study demonstrated that some important metabolic changes occurred in meropenem-induced CRKP-outer membrane vesicles (OMVs), The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-induced Klebsiella pneumoniae. Furthermore, this is the first study to discuss the protein-protein interaction network of the OMVs released by CRKP, especially under antibiotic stress.

Optimizing Conditions for the Production of Bacterial Extracellular Vesicles of Vibrio vulnificus and Analysis of the Inner Small RNA Compositions.

Chemical and physical elements affecting the production of bacterial extracellular vesicles (BEVs) of the human pathogen Vibrio vulnificus were quantitatively assessed to optimize the conditions for the BEV production by using the western blot quantification for an outer membrane porin OmpU and by fluorescent dye FM4-64. When cells were cultured at 37°C in an enriched medium (2 × Luria Bertani; 2 × LB) in the presence of EDTA, they produced about 70% more BEVs. BEVs were purified from the cells cultured in the established optimal conditions by the density gradient ultracentrifugation. The dynamic light scattering measurement of the purified BEVs showed that the diameter of them ranged from approximately 25 nm to 161 nm. We hypothesized that there may be some features in nucleotide sequences specific to RNAs packaged in BEVs compared to those in cellular RNA molecules. We compared the nucleotide sequences and abundance of sRNAs between in the cellular fraction and in BEVs through next-generation sequencing (NGS). While no distinct feature was observed in the nucleotide sequences of sRNAs between two groups, the length of sRNA fragments from BEVs were significantly shorter than those in cytoplasm.

Changes in the Murine Microbiome and Bacterial Extracellular Vesicle Production in Response to Antibiotic Treatment and Norovirus Infection.

Norovirus infection is influenced by the presence of commensal bacteria, and both human and murine norovirus (MNV) bind to these bacteria. These virus-bacterial interactions, as well as MNV infection, promote the increased production of bacterial extracellular vesicles (bEVs). However, no correlation has been made between specific bacterial groups, their vesicles, and their impact on norovirus infection. The current study evaluated the impact of select bacterial compositions of murine microbiomes using antibiotic (ABX) cocktails on MNV infection and bEV production. The goal of this research was to determine if increases in bEVs following MNV infection in mice were associated with changes in specific bacterial populations. Bacterial taxa were found to be differentially abundant in both ABX-treated and untreated mice, with the greatest change in bacterial taxa seen in mice treated with a broad-spectrum ABX cocktail. Specifically, Lachnospiraeae were found to be differentially abundant between a variety of treatment factors, including MNV infection. Overall, these results demonstrate that MNV infection can alter the abundance of bacterial taxa within the microbiota, as well as their production of extracellular vesicles, and that the use of selective antibiotic treatments can allow the detection of viral impacts on the microbiome that might otherwise be masked.

Elizabethkingia anophelis outer membrane vesicles as a novel vaccine candidate against infection: insights into immune response and potential for passive immunity.

IMPORTANCE: Elizabethkingia anophelis, a Gram-negative pathogen, causes infections such as bacteraemia, pneumonia, and neonatal meningitis. The pathogen resists most antimicrobial classes, making novel approaches urgently needed. In natural settings, Gram-negative bacteria secrete outer membrane vesicles (OMVs) that carry important molecules in the bacterial life cycle. These OMVs are enriched with proteins involved in virulence, survival, and carbohydrate metabolism, making them a promising source for vaccine development against the pathogen. This study investigated the efficacy of imipenem-induced OMVs (iOMVs) as a vaccine candidate against E. anophelis infection in a mouse pneumonia model. Mice immunized with iOMVs were completely protected during lethal-dose challenges. Passive immunization with hyperimmune sera and splenocytes conferred protection against lethal pneumonia. Further investigation is needed to understand the mechanisms underlying the protective effects of iOMV-induced passive immunity, such as the action on specific antibody subclasses or T cell subsets.