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Antimicrobial Resistance (AMR) has evolved from a mere concern into a significant global threat, with profound implications for public health, healthcare systems, and the global economy. Since the introduction of antibiotics between 1945 and 1963, their widespread and often indiscriminate use in human medicine, agriculture, and animal husbandry has led to the emergence and rapid spread of antibiotic-resistant genes. Bacteria have developed sophisticated mechanisms to evade the effects of antibiotics, including drug uptake limitation, drug degradation, target modification, efflux pumps, biofilm formation, and outer membrane vesicles production. As a result, AMR now poses a threat comparable to climate change and the COVID-19 pandemic, and projections suggest that death rates will be up to 10 million deaths annually by 2050, along with a staggering economic cost exceeding $100 trillion. Addressing AMR requires a multifaceted approach, including the development of new antibiotics, alternative therapies, and a significant shift in antibiotic usage and regulation. Enhancing global surveillance systems, increasing public awareness, and prioritizing investments in research, diagnostics, and vaccines are critical steps. By recognizing the gravity of the AMR threat and committing to collaborative action, its impact can be mitigated, and global health can be protected for future generations. [ABSTRACT FROM AUTHOR]

Bacterial infections, especially those caused by multidrug‐resistant pathogens, pose a significant threat to public health. Vaccines are a crucial tool in fighting these infections; however, no clinically available vaccine exists for the most common bacterial infections, such as those caused by Pseudomonas aeruginosa. Herein, a multiantigenic antibacterial nanovaccine (AuNP@HMV@SPs) is reported to combat P. aeruginosa infections. This nanovaccine utilizes the hybrid membrane vesicles (HMVs) created by fusing macrophage membrane vesicles (MMVs) with bacterial outer membrane vesicles (OMVs). The HMVs mitigate the toxic effects of both OMVs and bacterial secreted toxins (SP) adsorbed on the surface of MMVs, while preserving their stimulating properties. Gold nanoparticles (AuNPs) are utilized as adjuvant to enhance immune response without comprising safety. The nanovaccine AuNP@HMV@SPs induces robust humoral and cellular immune responses, leading to destruction of bacterial cells and neutralization of their secreted toxins. In murine models of septicemia and pneumonia caused by P. aeruginosa, AuNP@HMV@SPs exhibits superior prophylactic efficacy compared to control groups including OMVs, or MMVs@SPs and HMV@SPs, achieving 100% survival in septicemia and > 99.9% reduction in lung bacterial load in pneumonia. This study highlights AuNP@HMV@SPs as a safe and effective antibacterial nanovaccine, targeting both bacteria and their secreted toxins, and offers a promising platform for developing multiantigenic antibacterial vaccines against multidrug‐resistant pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) is a bacterium known to be associated with a significant risk of gastric cancer in addition to chronic gastritis, peptic ulcer, and MALT lymphoma. Although only a small percentage of patients infected with H. pylori develop gastric cancer, Gastric cancer causes more than 750,000 deaths worldwide, with 90% of cases being caused by H. pylori. The eradication of this bacterium rests on multiple drug regimens as guided by various consensus. However, the efficacy of empirical therapy is decreasing due to antimicrobial resistance. In addition, biofilm formation complicates eradication. As the search for new antibiotics lags behind the bacterium's ability to mutate, studies have been directed toward finding new anti-H. pylori agents while also optimizing current drug functions. Targeting biofilm, repurposing outer membrane vesicles that were initially a virulence factor of the bacteria, phage therapy, probiotics, and the construction of nanoparticles might be able to complement or even be alternatives for H. pylori treatment. This review aims to present reports on various compounds, either new or combined with current antibiotics, and their pathways to counteract H. pylori resistance. [ABSTRACT FROM AUTHOR]

With the coming of the post‐antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non‐replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live‐attenuated vaccines. Here, we developed a novel nano‐vaccine by coating OMVs onto PEGylated nano‐Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL‐OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL‐OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial‐specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T‐cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV‐coated nano‐carriers in antimicrobial immunotherapy. [ABSTRACT FROM AUTHOR]

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 (mreBR3) 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 mreBR3 showed eightfold higher vesicle production than the WT. ΔrodZ and mreBR3 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 mreBR3 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. [ABSTRACT FROM AUTHOR]

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 MnO 2 @OxA@OMV nanoreactor, enclosing bacterial-derived outer membrane vesicles (OMVs)-wrapped MnO 2 nanoenzyme and the immunogenic cell death inducer oxaliplatin (OxA), demonstrated intrinsic catalase-like activity within the TME, which effectively catalyzed the endogenous H 2 O 2 into O 2 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. A tailor-made nanoreactor was fabricated by enclosing bacterial-derived outer membrane vesicles (OMVs) onto MnO 2 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 H 2 O 2 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Bacterial infections, especially those caused by multidrug‐resistant pathogens, pose a significant threat to public health. Vaccines are a crucial tool in fighting these infections; however, no clinically available vaccine exists for the most common bacterial infections, such as those caused by Pseudomonas aeruginosa. Herein, a multiantigenic antibacterial nanovaccine (AuNP@HMV@SPs) is reported to combat P. aeruginosa infections. This nanovaccine utilizes the hybrid membrane vesicles (HMVs) created by fusing macrophage membrane vesicles (MMVs) with bacterial outer membrane vesicles (OMVs). The HMVs mitigate the toxic effects of both OMVs and bacterial secreted toxins (SP) adsorbed on the surface of MMVs, while preserving their stimulating properties. Gold nanoparticles (AuNPs) are utilized as adjuvant to enhance immune response without comprising safety. The nanovaccine AuNP@HMV@SPs induces robust humoral and cellular immune responses, leading to destruction of bacterial cells and neutralization of their secreted toxins. In murine models of septicemia and pneumonia caused by P. aeruginosa, AuNP@HMV@SPs exhibits superior prophylactic efficacy compared to control groups including OMVs, or MMVs@SPs and HMV@SPs, achieving 100% survival in septicemia and > 99.9% reduction in lung bacterial load in pneumonia. This study highlights AuNP@HMV@SPs as a safe and effective antibacterial nanovaccine, targeting both bacteria and their secreted toxins, and offers a promising platform for developing multiantigenic antibacterial vaccines against multidrug‐resistant pathogens.

Abstract With the coming of the post‐antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non‐replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live‐attenuated vaccines. Here, we developed a novel nano‐vaccine by coating OMVs onto PEGylated nano‐Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL‐OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL‐OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial‐specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T‐cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV‐coated nano‐carriers in antimicrobial immunotherapy.

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.

The highly multidrug-resistant (MDR) Gram-negative bacterial pathogen Acinetobacter baumannii is a top global health priority where an effective vaccine could protect susceptible populations and limit resistance acquisition. Outer membrane vesicles (OMVs) shed from Gram-negative bacteria are enriched with virulence factors and membrane lipids but heterogeneous in size and cargo. We report a vaccine platform combining precise and replicable nanoparticle technology with immunogenic A. baumannii OMVs (Ab-OMVs). Gold nanoparticle cores coated with Ab-OMVs (Ab-NPs) induced robust IgG titers in rabbits that enhanced human neutrophil opsonophagocytic killing and passively protected against lethal A. baumannii sepsis in mice. Active Ab-NP immunization in mice protected against sepsis and pneumonia, accompanied by B cell recruitment to draining lymph nodes, activation of dendritic cell markers, improved splenic neutrophil responses, and mitigation of proinflammatory cytokine storm. Nanoparticles are an efficient and efficacious platform for OMV vaccine delivery against A. baumannii and perhaps other high-priority MDR pathogens.

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. [ABSTRACT FROM AUTHOR]

Vaccination has become an increasingly attractive strategy for protecting against antibiotic-resistant infections. Nanovaccines based on the outer membrane from Gram-negative bacteria are appealing due to their multiantigenic nature and inherent immunogenicity. Here, we develop cellular nanodiscs made of bacterial outer membrane (OM-NDs), as a platform for antibacterial vaccination. Using Pseudomonas aeruginosa as a model pathogen, the resulting OM-NDs can effectively interact with antigen-presenting cells, exhibiting accelerated uptake and an improved capacity for immune stimulation. With their small size, the OM-NDs are also capable of efficiently transporting to the lymph nodes after in vivo administration. As a result, the nanovaccine is effective at eliciting potent humoral and cellular immune responses against P. aeruginosa. In a murine model of pneumonia, immunization with OM-NDs confers strong protection against subsequent lung infection, resulting in improved survival, reduced bacterial loads, and alleviation of immune overactivation. Overall, this report illustrates the advantages of cellular nanodiscs, which can be readily generalized to other pathogens and may be applied toward other biomedical applications.

Antimicrobial Resistance (AMR) has evolved from a mere concern into a significant global threat, with profound implications for public health, healthcare systems, and the global economy. Since the introduction of antibiotics between 1945 and 1963, their widespread and often indiscriminate use in human medicine, agriculture, and animal husbandry has led to the emergence and rapid spread of antibiotic-resistant genes. Bacteria have developed sophisticated mechanisms to evade the effects of antibiotics, including drug uptake limitation, drug degradation, target modification, efflux pumps, biofilm formation, and outer membrane vesicles production. As a result, AMR now poses a threat comparable to climate change and the COVID-19 pandemic, and projections suggest that death rates will be up to 10 million deaths annually by 2050, along with a staggering economic cost exceeding $100 trillion. Addressing AMR requires a multifaceted approach, including the development of new antibiotics, alternative therapies, and a significant shift in antibiotic usage and regulation. Enhancing global surveillance systems, increasing public awareness, and prioritizing investments in research, diagnostics, and vaccines are critical steps. By recognizing the gravity of the AMR threat and committing to collaborative action, its impact can be mitigated, and global health can be protected for future generations.

Helicobacter pylori (H. pylori) is a bacterium known to be associated with a significant risk of gastric cancer in addition to chronic gastritis, peptic ulcer, and MALT lymphoma. Although only a small percentage of patients infected with H. pylori develop gastric cancer, Gastric cancer causes more than 750,000 deaths worldwide, with 90% of cases being caused by H. pylori. The eradication of this bacterium rests on multiple drug regimens as guided by various consensus. However, the efficacy of empirical therapy is decreasing due to antimicrobial resistance. In addition, biofilm formation complicates eradication. As the search for new antibiotics lags behind the bacterium’s ability to mutate, studies have been directed toward finding new anti-H. pylori agents while also optimizing current drug functions. Targeting biofilm, repurposing outer membrane vesicles that were initially a virulence factor of the bacteria, phage therapy, probiotics, and the construction of nanoparticles might be able to complement or even be alternatives for H. pylori treatment. This review aims to present reports on various compounds, either new or combined with current antibiotics, and their pathways to counteract H. pylori resistance.

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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Various generations of membrane vesicles (MV) have been observed in Escherichia coli in terms of triggering events and populations of single-layered (s)/multi-layered (m) forms. Previously, we proposed a novel mechanism for MV generation triggered by the intracellular accumulation of biopolyester polyhydroxybutyrate (PHB). This was designated as the Polymer Intracellular Accumulation-triggered system for Membrane Vesicle Production (PIA-MVP). Herein, we attempted to determine the conditions for the change in the population between s-MV and m-MV using glucose concentration-dependent PIA-MVP. PIA-MVP was established using the good correlation between the glucose concentration-dependent PHB accumulation and MV generation. Thus, we assumed the presence of a critical glucose concentration could determine the population ratio of s-MV to m-MV, indicating that s-MV generation is a dominant component in the extracellular environment. Cytoplasmic green fluorescent protein (GFP) was used to evaluate the glucose concentration, enabling the selective generation of s-MV. The glucose concentration was determined to be 15 g/L to satisfy this purpose under the culture conditions. In conclusion, we established a biological system allowing us to selectively generate both single- and multi-layered MVs based on PIA-VIP encapsulation of GFP, providing a versatile toolkit to gain insights into the MV generation mechanism and achieve progress in various engineering applications.

Haoqian Chen,1 Xiao Zheng,2,3 Lingjue Li,1 Lishuxin Huang,1 Weiwei Huang,2 Yanbing Ma2 1Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, School of Ethnic Medicine, Yunnan Minzu University, Kunming, People’s Republic of China; 2Laboratory of Molecular Immunology, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, People’s Republic of China; 3School of Life Sciences, Yunnan University, Kunming, People’s Republic of ChinaCorrespondence: Lishuxin Huang, Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, School of Ethnic Medicine, Yunnan Minzu University, Kunming, 650504, People’s Republic of China, Tel/Fax +86 871 6592 6040, Email huanglishuxin19@163.com Yanbing Ma, Laboratory of Molecular Immunology, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiaoling Road, Kunming, 650032, People’s Republic of China, Tel +86 871 6833 9287, Fax +86 871 6833 4483, Email yanbingma1969@126.comBackground: Peptide-based vaccines have broad application prospects because of their safety, simple preparation, and effectiveness, especially in the development of personalized cancer vaccines, which have shown great advantages. However, the current peptide-based vaccines often require artificial synthesis and intricate delivery technology, which increases the cost and complexity of preparation.Methods: Here, we developed a simple technique for combining a peptide and a delivery system using the natural secretion system of bacteria. Specifically, we biosynthesized an antigenic peptide in bacteria, which was then extracellularly released through the bacterial secretory vesicles, thus simultaneously achieving the biosynthesis and delivery of the peptide.Results: The system utilizes the natural properties of bacterial vesicles to promote antigen uptake and dendritic cell (DC) maturation. Therefore, tumor-specific CD4+ Th1 and CD8+ cytotoxic T lymphocyte (CTL) responses were induced in TC-1 tumor-bearing mice, thereby efficiently suppressing tumor growth.Conclusion: This research promotes innovation and extends the application of peptide-based vaccine biosynthesis technology. Importantly, it provides a new method for personalized cancer immunotherapy that uses screened peptides as antigens in the future.Keywords: tumor immunization, peptide vaccine, outer membrane vesicle, delivery system

ABSTRACTCarbapenem-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.

ABSTRACTThe emerging pathogen Elizabethkingia anophelis poses severe threats to public health and has caused several outbreaks. The efficacy of antimicrobials is limited due to the pathogen’s innate multi-drug resistance to most antibiotics. Hence, novel approaches to treat E. anophelis infections are urgently needed. This study evaluated the immunogenic and protective effects of the imipenem-induced outer membrane vesicles (iOMVs) of E. anophelis as a vaccine. Mice immunized with iOMVs were completely protected during lethal-dose challenges. Passive immunization with hyperimmune sera and splenocytes conferred protection against lethal pneumonia. iOMVs also induced Th1-, Th2-, and Th17-driven immune responses. Immunization with iOMVs increased IgG1 and IgG2c isotype levels, and the antisera from immunized mice promoted complement-dependent bactericidal and opsonophagocytic activities against E. anophelis C08. Our study suggests that E. anophelis iOMVs are a promising vaccine candidate for preventing and treating E. anophelis infections and provides insights into the use of OMVs in vaccine development against the clinically emergent E. anophelis.IMPORTANCEElizabethkingia 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.

Objective: Porphyromonas gingivalis (Pg) is thought to be involved in the progression of Alzheimer's disease (AD). Whether Pg or its contents can reach the brain and directly affect neuropathology is, however, unknown. Here, we investigated whether outer membrane vesicles (OMVs) of Pg translocate to the brain and induce the pathogenic features of AD. Material and Methods: Pg OMVs were injected into the abdominal cavity of mice for 12 weeks. Pg OMV translocation to the brain was detected by immunohistochemistry using an anti‐gingipain antibody. Tau protein and microglial activation in the mouse brain were examined by western blotting and immunohistochemistry. The effect of gingipains on inflammation was assessed by real‐time polymerase chain reaction using human microglial HMC3 cells. Results: Gingipains were detected in the region around cerebral ventricles, choroid plexus, and ventricular ependymal cells in Pg OMV‐administered mice. Tau and phosphorylated Tau protein increased and microglia were activated. Pg OMVs also increased the gene expression of proinflammatory cytokines in HMC3 cells in a gingipain‐dependent manner. Conclusion: Pg OMVs, including gingipains, can reach the cerebral ventricle and induce neuroinflammation by activating microglia. Pg OMVs may provide a better understanding of the implications of periodontal diseases in neurodegenerative conditions such as AD. [ABSTRACT FROM AUTHOR]

Outer membrane vesicle (OMV)-delivered Pseudomonas quinolone signal (PQS) plays a critical role in cell-cell communication in Pseudomonas aeruginosa. However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from P. aeruginosa and liposome reduces the competitiveness of Burkholderia cenocepacia, which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in B. cenocepacia through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in B. cenocepacia. Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community. [ABSTRACT FROM AUTHOR]

Various generations of membrane vesicles (MV) have been observed in Escherichia coli in terms of triggering events and populations of single-layered (s)/multi-layered (m) forms. Previously, we proposed a novel mechanism for MV generation triggered by the intracellular accumulation of biopolyester polyhydroxybutyrate (PHB). This was designated as the Polymer Intracellular Accumulation-triggered system for Membrane Vesicle Production (PIA-MVP). Herein, we attempted to determine the conditions for the change in the population between s-MV and m-MV using glucose concentration-dependent PIA-MVP. PIA-MVP was established using the good correlation between the glucose concentration-dependent PHB accumulation and MV generation. Thus, we assumed the presence of a critical glucose concentration could determine the population ratio of s-MV to m-MV, indicating that s-MV generation is a dominant component in the extracellular environment. Cytoplasmic green fluorescent protein (GFP) was used to evaluate the glucose concentration, enabling the selective generation of s-MV. The glucose concentration was determined to be 15 g/L to satisfy this purpose under the culture conditions. In conclusion, we established a biological system allowing us to selectively generate both single- and multi-layered MVs based on PIA-VIP encapsulation of GFP, providing a versatile toolkit to gain insights into the MV generation mechanism and achieve progress in various engineering applications. [ABSTRACT FROM AUTHOR]

Abstract Bacteria form a highly complex ecosystem in the gastrointestinal (GI) tract. In recent years, mounting evidence has shown that bacteria can release nanoscale phospholipid bilayer particles that encapsulate nucleic acids, proteins, lipids, and other molecules. Extracellular vesicles (EVs) are secreted by microorganisms and can transport a variety of important factors, such as virulence factors, antibiotics, HGT, and defensive factors produced by host eukaryotic cells. In addition, these EVs are vital in facilitating communication between microbiota and the host. Therefore, bacterial EVs play a crucial role in maintaining the GI tract’s health and proper functioning. In this review, we outlined the structure and composition of bacterial EVs. Additionally, we highlighted the critical role that bacterial EVs play in immune regulation and in maintaining the balance of the gut microbiota. To further elucidate progress in the field of intestinal research and to provide a reference for future EV studies, we also discussed the clinical and pharmacological potential of bacterial EVs, as well as the necessary efforts required to understand the mechanisms of interaction between bacterial EVs and gut pathogenesis.

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.

Rapid detection and quantification of outer membrane vesicle (OMV) are of both scientific value and clinical implications. However, limited tools are available for investigations of OMVs. Herein, we report a novel fluorescent probe with aggregation-induced emission (AIE) characteristics, namely, OEO-TPE-MEM (OTM), for OMV detection. OTM emits faintly in an aqueous medium, but its fluorescence could be effectively turned on upon interacting with bacteria bodies and OMVs produced by Gram-negative bacteria. Notably, OTM could provide quantitative information on bacterial membrane remodeling and OMV secretion and be applied to high-throughput screening of OMV-inducing agents. This study presents a powerful AIE probe for imaging and quantitative analysis of bacteria envelop and derived OMVs, which might be applied for evaluating research and clinical antimicrobial materials in future studies. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. Key points: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is a nosocomial pathogen and an important propagator of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Like other Gram-negative bacteria, they secrete outer membrane vesicles (OMVs) that distribute virulence and resistance factors. Here, we subjected a K. pneumoniae-XDR to subinhibitory concentrations of meropenem, amikacin, polymyxin B, and a combination of these agents to evaluate changes in the protein cargo of OMVs through liquid chromatography–tandem mass spectrometry (LC-MS/MS). Genome sequencing of the clinical isolate K. pneumoniae strain HCD1 (KpHCD1) revealed the presence of 41 resistance genes and 159 virulence factors. We identified 64 proteins in KpHCD1-OMVs modulated with different antibiotic treatments involved in processing genetic information, environmental information, cell envelope formation, energy metabolism, and drug resistance. The OMV proteome expression profile suggests that OMVs may be associated with pathogenicity, survival, stress response, and resistance dissemination. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the causative agent of a respiratory infection called pertussis (whooping cough) that can be fatal in newborns and infants. The pathogen produces a variety of antigenic compounds which alone or simultaneously can damage various host cells. Despite the availability of pertussis vaccines and high vaccination coverage around the world, a resurgence of the disease has been observed in many countries. Reasons for the increase in pertussis cases may include increased awareness, improved diagnostic techniques, low vaccine efficacy, especially acellular vaccines, and waning immunity. Many efforts have been made to develop more effective strategies to fight against B. pertussis and one of the strategies is the use of outer membrane vesicles (OMVs) in vaccine formulations. OMVs are attracting great interest as vaccine platforms since they can carry immunogenic structures such as toxins and LPS. Many studies have been carried out with OMVs from different B. pertussis strains and they revealed promising results in the animal challenge and human preclinical model. However, the composition of OMVs differs in terms of isolation and purification methods, strains, culture, and stress conditions. Although the vesicles from B. pertussis represent an attractive pertussis vaccine candidate, further studies are needed to advance clinical research for next-generation pertussis vaccines. This review summarizes general information about pertussis, the history of vaccines against the disease, and the immune response to these vaccines, with a focus on OMVs. We discuss progress in developing an OMV-based pertussis vaccine platform and highlight successful applications as well as potential challenges and gaps. [ABSTRACT FROM AUTHOR]

Abstract The role of gut microbiota and its products in human health and disease is profoundly investigated. The communication between gut microbiota and the host involves a complicated network of signaling pathways via biologically active molecules generated by intestinal microbiota. Some of these molecules could be assembled within nanoparticles known as outer membrane vesicles (OMVs). Recent studies propose that OMVs play a critical role in shaping immune responses, including homeostasis and acute inflammatory responses. Moreover, these OMVs have an immense capacity to be applied in medical research, such as OMV-based vaccines and drug delivery. This review presents a comprehensive overview of emerging knowledge about biogenesis, the role, and application of these bacterial-derived OMVs, including OMV-based vaccines, OMV adjuvants characteristics, OMV vehicles (in conjugated vaccines), cancer immunotherapy, and drug carriers and delivery systems. Moreover, we also highlight the significance of the potential role of these OMVs in diagnosis and therapy.

Extracellular vesicles (EVs) are secreted by both eukaryotes and prokaryotes, and are present in all biological fluids of vertebrates, where they transfer DNA, RNA, proteins, lipids, and metabolites from donor to recipient cells in cell-to-cell communication. Some EV components can also indicate the type and biological status of their parent cells and serve as diagnostic targets for liquid biopsy. EVs can also natively carry or be modified to contain therapeutic agents (e.g., nucleic acids, proteins, polysaccharides, and small molecules) by physical, chemical, or bioengineering strategies. Due to their excellent biocompatibility and stability, EVs are ideal nanocarriers for bioactive ingredients to induce signal transduction, immunoregulation, or other therapeutic effects, which can be targeted to specific cell types. Herein, we review EV classification, intercellular communication, isolation, and characterization strategies as they apply to EV therapeutics. This review focuses on recent advances in EV applications as therapeutic carriers from in vitro research towards in vivo animal models and early clinical applications, using representative examples in the fields of cancer chemotherapeutic drug, cancer vaccine, infectious disease vaccines, regenerative medicine and gene therapy. Finally, we discuss current challenges for EV therapeutics and their future development.

Bacteria form a highly complex ecosystem in the gastrointestinal (GI) tract. In recent years, mounting evidence has shown that bacteria can release nanoscale phospholipid bilayer particles that encapsulate nucleic acids, proteins, lipids, and other molecules. Extracellular vesicles (EVs) are secreted by microorganisms and can transport a variety of important factors, such as virulence factors, antibiotics, HGT, and defensive factors produced by host eukaryotic cells. In addition, these EVs are vital in facilitating communication between microbiota and the host. Therefore, bacterial EVs play a crucial role in maintaining the GI tract's health and proper functioning. In this review, we outlined the structure and composition of bacterial EVs. Additionally, we highlighted the critical role that bacterial EVs play in immune regulation and in maintaining the balance of the gut microbiota. To further elucidate progress in the field of intestinal research and to provide a reference for future EV studies, we also discussed the clinical and pharmacological potential of bacterial EVs, as well as the necessary efforts required to understand the mechanisms of interaction between bacterial EVs and gut pathogenesis. [ABSTRACT FROM AUTHOR]

Predatory outer membrane vesicles (OMVs) secreted by myxobacteria fuse readily with the outer membranes of Gram-negative bacteria, introducing toxic cargo into their prey. Here we used a strain of the myxobacterium Myxococcus xanthus that produces fluorescent OMVs to assay the uptake of OMVs by a panel of Gram-negative bacteria. M. xanthus strains took up significantly less OMV material than the tested prey strains, suggesting that re-fusion of OMVs with producing organisms is somehow inhibited. The OMV killing activity against different prey correlated strongly with the predatory activity of myxobacterial cells, however, there was no correlation between OMV killing activity and their propensity to fuse with different prey. It has previously been proposed that M. xanthus GAPDH stimulates the predatory activity of OMVs by enhancing OMV fusion with prey cells. Therefore, we expressed and purified active fusion proteins of M. xanthus glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (GAPDH and PGK; moonlighting enzymes with additional activities beyond their roles in glycolysis/gluconeogenesis) to investigate any involvement in OMV-mediated predation. Neither GAPDH nor PGK caused lysis of prey cells or enhanced OMV-mediated lysis of prey cells. However, both enzymes were found to inhibit the growth of Escherichia coli, even in the absence of OMVs. Our results suggest that fusion efficiency is not a determinant of prey killing, but instead resistance to the cargo of OMVs and co-secreted enzymes dictates whether organisms can be preyed upon by myxobacteria. [ABSTRACT FROM AUTHOR]

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.

Outer membrane vesicles (OMVs) of bacteria harbor physiologically active molecules, and quorum sensing inhibitors (QSIs) are expected to regulate bacterial virulence. In this study, we analyzed the proinflammatory activity of OMVs of the periodontal pathogen Tannerella forsythia treated with d‐arabinose and d‐galactose as QSIs, which inhibit the biofilm formation of periodontal pathogens and autoinducer 2 activity. Compared to OMVs of nontreated T. forsythia (TF OMVs), OMVs released from QSI‐treated T. forsythia, designated TF ara‐OMVs and TF gal‐OMVs, showed reduced production of TNF‐α, IL‐1β, IL‐6, and IL‐8 in THP‐1 monocytes through decreased activation of NF‐κB/MAPKs. Using a human NF‐κB reporter cell line and bone marrow‐derived macrophages from TLR2−/− mice, TF ara‐OMVs and TF gal‐OMVs showed less activation of TLR2 than TF OMVs. These results demonstrated that QSIs provide a dual advantage against bacterial infection by inhibiting bacterial biofilm formation and generating OMVs with reduced proinflammatory activity. [ABSTRACT FROM AUTHOR]

The role of gut microbiota and its products in human health and disease is profoundly investigated. The communication between gut microbiota and the host involves a complicated network of signaling pathways via biologically active molecules generated by intestinal microbiota. Some of these molecules could be assembled within nanoparticles known as outer membrane vesicles (OMVs). Recent studies propose that OMVs play a critical role in shaping immune responses, including homeostasis and acute inflammatory responses. Moreover, these OMVs have an immense capacity to be applied in medical research, such as OMV-based vaccines and drug delivery. This review presents a comprehensive overview of emerging knowledge about biogenesis, the role, and application of these bacterial-derived OMVs, including OMV-based vaccines, OMV adjuvants characteristics, OMV vehicles (in conjugated vaccines), cancer immunotherapy, and drug carriers and delivery systems. Moreover, we also highlight the significance of the potential role of these OMVs in diagnosis and therapy. [ABSTRACT FROM AUTHOR]

BACKGROUND: Extracellular vesicles can control the inflammatory response by participating in immunomodulation of intercellular signaling, while also repairing periodontal tissue and promoting periodontal tissue regeneration. The application of this technology to the preventive diagnosis and tissue regeneration of chronic periodontitis is expected to become a method of cell-free tissue regeneration. OBJECTIVE: To review the synthesis and secretion of extracellular vesicles, biological function, relationship with chronic periodontitis, and recent advances in the diagnosis, prevention and treatment of chronic periodontitis. METHODS: The first author applied the computer to search for relevant studies involving extracellular vesicles in the prevention and treatment of chronic periodontitis in databases such as PubMed, Web of Science and CNKI. The search keywords were “extracellular vesicle, exosome, periodontal disease, immunomodulatory, outermembrane vesicles, bone regeneration” in Chinese and English. The retrieval time was from April 2014 to October 2021. RESULTS AND CONCLUSION: Extracellular vesicles are balloon-like bodies that can be produced by almost all cells, which are widely present in various body fluids, and are rich in proteins, lipids and genetic material on the surface, which affect the biological behavior of target cells by mediating information transfer between cells. On the one hand, the inflammatory response of chronic periodontitis is inseparable from the mediation of extracellular vesicles secreted by bacteria, which can be used as a biomarker to diagnose and evaluate chronic periodontitis; on the other hand, the new chronic periodontitis vaccine made by extracellular vesicles has initially been effective, and the extracellular vesicles containing information molecules can also participate in regulating the immune response and promoting the repair and regeneration of periodontal tissue. Therefore, in the field of tissue damage repair and regeneration of chronic periodontitis, extracellular vesicles are a new and potential cell-free therapy, but their extraction, purification and safety are still challenges to be overcome before clinical translation. [ABSTRACT FROM AUTHOR]

Bacterial outer membrane vesicles (OMVs) are 20- to 200-nm secreted packages of lipids, small molecules, and proteins that contribute to diverse bacterial processes. In plant systems, OMVs from pathogenic and beneficial strains elicit plant immune responses that inhibit seedling growth and protect against future pathogen challenge. Previous studies of OMV-plant interactions suggest functionally important differences in the protein composition of Pseudomonas syringae and Pseudomonas fluorescens OMVs, and that their composition and activity differ as a result of medium culture conditions. Here, we show that plant apoplast-mimicking minimal medium conditions impact OMV protein content dramatically in P. syringae but not in P. fluorescens relative to complete medium conditions. Comparative, 2-way analysis of the four conditions reveals subsets of proteins that may contribute to OMV-mediated bacterial virulence and plant immune activation as well as those involved in bacterial stress tolerance or adaptation to a beneficial relationship with plants. Additional localization enrichment analysis of these subsets suggests the presence of outer-inner membrane vesicles (OIMVs). Collectively, these results reveal distinct differences in bacterial extracellular vesicle cargo and biogenesis routes from pathogenic and beneficial plant bacteria in different medium conditions and point to distinct populations of vesicles with diverse functional roles. [ABSTRACT FROM AUTHOR]

Escherichia coli strains are responsible for a majority of human extra-intestinal infections, resulting in huge direct medical and social costs. We had previously shown that HlyF encoded by a large virulence plasmid harbored by pathogenic E. coli is not a hemolysin but a cytoplasmic enzyme leading to the overproduction of outer membrane vesicles (OMVs). Here, we showed that these specific OMVs inhibit the macroautophagic/autophagic flux by impairing the autophagosome-lysosome fusion, thus preventing the formation of acidic autolysosomes and autophagosome clearance. Furthermore, HlyF-associated OMVs were more prone to activate the non-canonical inflammasome pathway. Because autophagy and inflammation are crucial in the host's response to infection especially during sepsis, our findings revealed an unsuspected role of OMVs in the crosstalk between bacteria and their host, highlighting the fact that these extracellular vesicles have exacerbated pathogenic properties. Abbreviations: AIEC: adherent-invasive E. coliBDI: bright detail intensityBMDM: bone marrow‐derived macrophagesCASP: caspaseE. coli: Escherichia coliEHEC: enterohemorrhagic E. coliExPEC: extra-intestinal pathogenic E. coliGSDMD: gasdermin DGFP: green fluorescent proteinHBSS: Hanks' balanced salt solutionHlyF: hemolysin FIL1B/IL‐1B: interleukin 1 betaISX: ImageStreamX systemLPS: lipopolysaccharideMut: mutatedOMV: outer membrane vesicleRFP: red fluorescent proteinTEM: transmission electron microscopyWT: wild-type [ABSTRACT FROM AUTHOR]

The highly multidrug‐resistant (MDR) Gram‐negative bacterial pathogen Acinetobacter baumannii is a top global health priority where an effective vaccine can protect susceptible populations and limit resistance acquisition. Outer membrane vesicles (OMVs) shed from Gram‐negative bacteria are enriched with virulence factors and membrane lipids but heterogeneous in size and cargo. Herein, a vaccine platform combining precise and replicable nanoparticle technology with immunogenic A. baumannii OMVs (Ab‐OMVs) is reported. Gold nanoparticle cores coated with Ab‐OMVs (Ab‐NPs) induce robust IgG titers in rabbits that enhance human neutrophil opsonophagocytic killing and passively protect against lethal A. baumannii sepsis in mice. Active Ab‐NP immunization in mice protects against sepsis and pneumonia, accompanied by B cell recruitment to draining lymph nodes, activation of dendritic cell markers, improved splenic neutrophil responses, and mitigation of proinflammatory cytokine storm. Nanoparticles are an efficient and efficacious platform for OMV vaccine delivery against A. baumannii and perhaps other high‐priority MDR pathogens.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the β-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Outer membrane vesicles (OMVs) are non-living spherical nanostructures that derive from the cell envelope of Gram-negative bacteria. OMVs are important in bacterial pathogenesis, cell-to-cell communication, horizontal gene transfer, quorum sensing, and in maintaining bacterial fitness. These structures can be modified to express antigens of interest using glycoengineering and genetic or chemical modification. The resulting OMVs can be used to immunize individuals against the expressed homo- or heterologous antigens. Additionally, cargo can be loaded into OMVs and they could be used as a drug delivery system. OMVs are inherently immunogenic due to proteins and glycans found on Gram negative bacterial outer membranes. This review focuses on OMV manipulation to increase vesiculation and decrease antigenicity, their utility as vaccines, and novel engineering approaches to extend their application.

Vaccine platforms enable fast development, testing, and manufacture of more affordable vaccines. Here, we evaluated Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles (OMVs) generated by genetically modified Gram‐negative bacteria, as a vaccine platform for viral pathogens. Influenza A virus hemagglutinin (HA), either physically mixed with GMMA (HA+STmGMMA mix), or covalently linked to GMMA surface (HA‐STmGMMA conjugate), significantly increased antigen‐specific humoral and cellular responses, with HA‐STmGMMA conjugate inducing further enhancement than HA+STmGMMA mix. HA‐STmGMMA conjugate protected mice from lethal challenge. The versatility for this platform was confirmed by conjugation of rabies glycoprotein (RABVG) onto GMMA through the same method. RABVG+STmGMMA mix and RABVG‐STmGMMA conjugate exhibited similar humoral and cellular response patterns and protection efficacy as the HA formulations, indicating relatively consistent responses for different vaccines based on the GMMA platform. Comparing to soluble protein, GMMA was more efficiently taken up in vivo and exhibited a B‐cell preferential uptake in the draining lymph nodes (LNs). Together, GMMA enhances immunity against viral antigens, and the platform works well with different antigens while retaining similar immunomodulatory patterns. The findings of our study imply the great potential of GMMA‐based vaccine platform also against viral infectious diseases. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are secreted by both eukaryotes and prokaryotes, and are present in all biological fluids of vertebrates, where they transfer DNA, RNA, proteins, lipids, and metabolites from donor to recipient cells in cell-to-cell communication. Some EV components can also indicate the type and biological status of their parent cells and serve as diagnostic targets for liquid biopsy. EVs can also natively carry or be modified to contain therapeutic agents (e.g. , nucleic acids, proteins, polysaccharides, and small molecules) by physical, chemical, or bioengineering strategies. Due to their excellent biocompatibility and stability, EVs are ideal nanocarriers for bioactive ingredients to induce signal transduction, immunoregulation, or other therapeutic effects, which can be targeted to specific cell types. Herein, we review EV classification, intercellular communication, isolation, and characterization strategies as they apply to EV therapeutics. This review focuses on recent advances in EV applications as therapeutic carriers from in vitro research towards in vivo animal models and early clinical applications, using representative examples in the fields of cancer chemotherapeutic drug, cancer vaccine, infectious disease vaccines, regenerative medicine and gene therapy. Finally, we discuss current challenges for EV therapeutics and their future development. Different sources' extracellular vesicles (EVs) can load various therapeutic agents to play the excellent therapeutic role in the fields of cancer therapy, infectious disease vaccines, regenerative medicine and gene therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Citrus Huanglongbing (HLB) is the most devastating citrus disease in the world. Candidatus Liberibacter asiaticus (Las) is the prevalent HLB pathogen, which is yet to be cultivated. A recent study demonstrates that Las does not contain pathogenicity factors that are directly responsible for HLB symptoms. Instead, Las triggers systemic and chronic immune responses, representing a pathogen-triggered immune disease. Importantly, overproduction of reactive oxygen species (ROS) causes systemic cell death of phloem tissues, thus causing HLB symptoms. Because Las resides in the phloem tissues, it is expected that phloem cell might recognize outer membrane proteins, outer membrane vesicle (OMV) proteins and extracellular proteins of Las to contribute to the immune responses. Because Las has not been cultivated, we used Liberibacter crescens (Lcr) as a surrogate to identify proteins in the OM fraction, OMV proteins and extracellular proteins by liquid chromatography with tandem mass spectrometry (LC–MS/MS). We observed OMVs of Lcr under scanning electron microscope, representing the first experimental evidence that Liberibacter can deliver proteins to the extracellular compartment. In addition, we also further analyzed LC–MS/MS data using bioinformatic tools. Our study provides valuable information regarding the biology of Ca. Liberibacter species and identifies many putative proteins that may interact with host proteins in the phloem tissues. [ABSTRACT FROM AUTHOR]

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., (© 2024 Wiley‐VCH GmbH.)

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., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)