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Numerous research studies have established a connection between hypervirulent Klebsiella pneumoniae (hvKP) and bovine abortion, as well as other complications and clinical illnesses. These bacteria, which belong to the Gram-negative group, release outer membrane vesicles (OMVs), believed to play a crucial role in transporting effector molecules to their target cells. To investigate this further, OMVs of hvKP were collected from 47 clinical isolates from bovine uterine discharge samples using ultracentrifugation. Confirmation of the 47 isolates was carried out through a standard PCR test targeting the 16S rRNA gene. Subsequently, a multiplex PCR was used to verify positive 16S rRNA gene results by detecting specific virulence genes (fimH, acrAB, traT, and rmpA). The standard PCR test showed that 27 out of the 47 isolates (57.45%) tested positive for the 16S rRNA gene, with all 27 of these isolates also testing positive for the rmpA and traT genes. Additionally, 30% of the isolates (8.1 out of 27) tested positive for AcrAB, while 70% of the isolates (18.9 out of 27) tested positive for FimH. During SDS-Western blotting, ten protein bands with molecular weights ranging from 18 to >60 kDa were identified in K. pneumoniae-derived OMV preparations. These bands included OmpX, flagellin, MipA, OmpA, OmpC, and OmpF (42-44 kDa), a strong OmpA band at 45 KDa, and a faint band at 60 kDa of GroEL as an oligomer of OmpA. Furthermore, hvKP OMVs displayed varying cytotoxic effects on the L929 and THP-1 cell lines. Notably, incubating THP-1 cells with high concentrations of hvKP OMVs inhibited IL-1β and TNF responses, indicating that these OMVs may not only facilitate the internalisation of the bacterium by THP-1 cells but also suppress the cells’ innate immune response to hvKP infection. This ability of hvKP OMVs to evade the immune system and modulate host responses contributes significantly to the bacterium’s pathogenicity. [ABSTRACT FROM AUTHOR]

Chromobacterium violaceum is a pathogenic bacterium that can infect humans and animals, yet the role of its outer membrane vesicles (OMVs) in mediating pathogenicity remains underexplored. This study evaluated the effects of linoleic acid (LA) and stearic acid (SA) on quorum sensing (QS)-mediated violacein production, biofilm formation, and OMV biogenesis in C. violaceum. Our findings revealed that 2 mM LA and 1 mM SA effectively quench QS, leading to a significant reduction in violacein production, biofilm formation, and OMV biogenesis. Gene expression analysis confirmed the downregulation of QS-related genes, including cviI, cviR, vioA, vioB, and vioC, in fatty acid-treated C. violaceum. Additionally, we assessed the antimicrobial activity of C. violaceum-derived OMVs on Rhizobium sp., a PGPR and observed a marked reduction in bactericidal activity in the treated OMVs. This study suggests that LA and SA have potential as anti-infective agents to mitigate OMV-mediated virulence and combat antibiotic resistance in pathogens. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigates the set of proteins that comprise a Vibrio ordalii strain Vo-LM-18 and its outer membrane vesicles (OMVs), which are involved in the pathogen's interaction with fish hosts, especially salmonids. Vibrio ordalii is a major cause of vibriosis, a disease that results in significant mortality in fish farms, a relevant industry in Chile. By analyzing the proteins expressed by both the bacteria and their OMVs, this research identifies key proteins linked to virulence, iron uptake, and cellular communication. The findings suggest that OMVs carry virulence factors that could aid in the pathogen's ability to infect and persist in fish. These vesicles may also contribute to the pathogen's survival in harsh environments, such as during host immune responses. This study provides new insights into bacterial pathogenesis and highlights potential targets for developing treatments or vaccines to combat infections in aquaculture. Vibrio ordalii is the causative agent of atypical vibriosis in salmonids cultured in Chile. While extensive research provides insights into V. ordalii through phenotypic, antigenic, and genetic typing, as well as various virulence mechanisms, proteomic characterization remains largely unexplored. This study aimed to advance the proteomic knowledge of Chilean V. ordalii Vo-LM-18 and its OMVs, which have known virulence. Using Nano-UHPLC-LC-MS/MS, we identified 2242 proteins and 1755 proteins in its OMVs. Of these, 644 unique proteins were detected in V. ordalii Vo-LM-18, namely 156 unique proteins in its OMVs and 1596 shared proteins. The major categories for the OMVs were like those in the bacteria (i.e., cytoplasmic and cytoplasmic membrane proteins). Functional annotation identified 37 biological pathways in V. ordalii Vo-LM-18 and 28 in its OMVs. Proteins associated with transport, transcription, and virulence were predominant in both. Evident differences in protein expression were found. OMVs expressed a higher number of virulence-associated proteins, including those related to iron- and heme-uptake mechanisms. Notable pathways in the bacteria included flagellum assembly, heme group-associated proteins, and protein biosynthesis. This proteomic analysis is the first to detect the RTX toxin in a V. ordalii strain (Vo-LM-18) and its vesicles. Our results highlight the crucial role of OMVs in the pathogenesis and adaptation of V. ordalii, suggesting use as potential diagnostic biomarkers and therapeutic targets for bacterial infections. [ABSTRACT FROM AUTHOR]

Teredinibacter turnerae is a cultivable cellulolytic Gammaproteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood‐eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose and pectin and contribute to wood (lignocellulose) digestion in the shipworm gut. However, the mechanisms by which T. turnerae secretes lignocellulolytic enzymes are incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce membrane vesicles (MVs) that include a variety of proteins identified by liquid chromatography–mass spectrometry (LC–MS/MS) as carbohydrate‐active enzymes (CAZymes) with predicted activities against cellulose, hemicellulose and pectin. Reducing sugar assays and zymography confirm that these MVs exhibit cellulolytic activity, as evidenced by the hydrolysis of CMC. Additionally, these MVs were enriched with TonB‐dependent receptors, which are essential to carbohydrate and iron acquisition by free‐living bacteria. These observations indicate a potential role for MVs in lignocellulose utilisation by T. turnerae in the free‐living state, suggest possible mechanisms for host–symbiont interaction and may be informative for commercial applications such as enzyme production and lignocellulosic biomass conversion. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) are extracellular structures, ranging in size from 10 to 300 nm, produced by Gram-negative bacteria. They can be incorporated into the outer membrane of a recipient's cells, which may enable the transfer of substances with lytic properties. Due to the scarce information regarding the OMVs produced by Proteus mirabilis, the aim of this study was to test the blebbing abilities of the clinical P. mirabilis O77 and O78 strains and to determine the blebs' interactions with bacterial cells, including their possible bactericidal activities. The production of OMVs was visualised by Transmission electron microscopy (TEM). The presence of OMVs in the obtained samples as well as the phenomenon of OMV fusion to recipient cells were confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting assays. The bacteriolytic activity of the OMVs was examined against P. mirabilis clinical strains and reference Staphylococcus aureus and Escherichia coli strains. It was shown that each of the two tested P. mirabilis strains could produce OMVs which were able to fuse into the cells of the other strain. The lytic properties of the O78 OMVs against another P. mirabilis O78 strain were also demonstrated. This promising result may help in the future to better understand the mechanisms of the pathogenesis and to treat the infections caused by P. mirabilis. [ABSTRACT FROM AUTHOR]

Oral pathobionts are essential in instigating local inflammation within the oral cavity and contribute to the pathogenesis of diseases in the gastrointestinal tract and other distant organs. Among the Gram-negative pathobionts, Porphyromonas gingivalis and Fusobacterium nucleatum emerge as critical drivers of periodontitis, exerting their influence not only locally but also as inducers of gut dysbiosis, intestinal disturbances, and systemic ailments. This dual impact is facilitated by their ectopic colonization of the intestinal mucosa and the subsequent mediation of distal systemic effects by releasing outer membrane vesicles (OMVs) into circulation. This review elucidates the principal components of oral pathobiont-derived OMVs implicated in disease pathogenesis within the oral–gut axis, detailing virulence factors that OMVs carry and their interactions with host epithelial and immune cells, both in vitro and in vivo. Additionally, we shed light on the less acknowledged interplay between oral pathobionts and the gut commensal Akkermansia muciniphila, which can directly impede oral pathobionts' growth and modulate bacterial gene expression. Notably, OMVs derived from A. muciniphila emerge as promoters of anti-inflammatory effects within the gastrointestinal and distant tissues. Consequently, we explore the potential of A. muciniphila-derived OMVs to interact with oral pathobionts and prevent disease in the oral–gut axis. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) are nano-sized vesicles actively released by Gram-negative bacteria, playing a crucial role in bacterial survival and interactions with phages. This review focuses on OMVs and succinctly delineates the stimuli instigating OMV formation, their functional repertoire, and their involvement in bacterial-phage interplays. Initially, the discussion centers on the drivers prompting OMV genesis, encompassing both extrinsic environmental pressures and intrinsic regulatory mechanisms within bacterial systems. Subsequently, a comprehensive examination of OMVs’ multifaceted functions in bacterial physiology ensues, spanning signaling cascades, nutrient transport, antibiotic resilience, and evasion of immune surveillance. Particular emphasis is placed on elucidating the paramount significance of OMVs in mediating bacterial-phage dynamics. OMVs function as decoys, providing protection to bacterial hosts against phages, and concurrently promoting the spread of phage receptors, thereby rendering phage-resistant strains susceptible to phage invasion. This comprehensive review deepens our comprehension of membrane vesicles biogenesis in bacteria and their pivotal role in microbial community dynamics. [ABSTRACT FROM AUTHOR]

Abstract CprA is a short‐chain dehydrogenase/reductase (SDR) that contributes to resistance against colistin and antimicrobial peptides. The cprA gene is conserved across Pseudomonas aeruginosa clades and its expression is directly regulated by the two‐component system PmrAB. We have shown that cprA expression leads to the production of outer membrane vesicles (OMVs) that block autophagic flux and have a greater capacity to activate the non‐canonical inflammasome pathway. In a murine model of sepsis, a P. aeruginosa strain deleted for cprA was less virulent than the wild‐type (WT) strain. These results demonstrate the important role of CprA in the pathogenicity of P. aeruginosa. It is worth noting that CprA is also a functional ortholog of hemolysin F (HlyF), which is encoded by virulence plasmids of Escherichia coli. We have shown that other cryptic SDRs encoded by mammalian and plant pathogens, such as Yersinia pestis and Ralstonia solanacearum are functional orthologs of CprA and HlyF. These SDRs also induce the production of OMVs which block autophagic flux. This study uncovers a new family of virulence determinants in Gram‐negative bacteria, offering potential for innovative therapeutic interventions and deeper insights into bacterial pathogenesis.

Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against A. baumannii, using both LOS-containing and LOS-free OMVs preparations. [ABSTRACT FROM AUTHOR]

ABSTRACT Inflammatory bowel diseases are characterized by chronic intestinal inflammation and alterations in the gut microbiota composition. Bacteroides fragilis, which secretes outer membrane vesicles (OMVs) with polysaccharide A (PSA), can moderate the inflammatory response and possibly alter the microbiota composition. In this study, we created a murine model of chronic sodium dextran sulfate (DSS)-induced intestinal colitis and treated it with B. fragilis OMVs. We monitored the efficiency of OMV therapy by determining the disease activity index (DAI) and performing histological examination (HE) of the intestine before and after vesicle exposure. We also analyzed the microbiota composition using 16S rRNA gene sequencing. Finally, we evaluated the volatile compound composition in the animals’ stools by HS-GC/MS to assess the functional activity of the microbiota. We observed more effective intestinal repair after OMV treatment according to the DAI and HE. A metabolomic study also revealed changes in the functional activity of the microbiota, with a predominance of phenol and pentanoic acid in the control group compared to the group treated with DSS and the group treated with OMVs (DSS OMVs). We also observed a positive correlation of these metabolites with Saccharibacteria and Acetivibrio in the control group, whereas in the DSS group, there was a negative correlation of phenol and pentanoic acid with Lactococcus and Romboutsia. According to the metabolome and sequencing data, the microbiota composition of the DSS-treated OMV group was intermediate between that of the control and DSS groups. OMVs not only have an anti-inflammatory effect but also contribute to the recovery of the microbiota composition.IMPORTANCEBacteroides fragilis vesicles contain superficially localized polysaccharide A (PSA), which has unique immune-modulating properties. Isolated PSA can prevent chemically induced colitis in a murine model. Outer membrane vesicles (OMVs) also contain digestive enzymes and volatile metabolites that can complement the anti-inflammatory properties of PSA. OMVs showed high therapeutic activity against sodium dextran sulfate-induced colitis, as confirmed by histological assays. 16S rRNA sequencing of fecal samples from different inflammatory stages, supplemented with comprehensive metabolome analysis of volatile compounds conducted by HS-GC/MS, revealed structural and functional alterations in the microbiota composition under the influence of OMVs. Correlation analysis of the OMV-treated and untreated experimental animal groups revealed associations of phenol and pentanoic acid with Lactococcus, Romboutsia, Saccharibacteria, and Acetivibrio.

Flavobacterium psychrophilum (Fp) causes Bacterial Cold Water Disease in salmonids. During host‐pathogen interactions, gram‐negative bacteria, such as Fp, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs' small RNAs (sRNAs) in the Fp‐rainbow trout host‐pathogen interactions. sRNAs carried within OMVs were isolated from Fp. RNA‐Seq datasets from whole‐cell Fp and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV‐packaged sRNAs were located in the pathogenicity islands of Fp. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA‐Seq of host and pathogen transcriptomes on day 5 post‐infection of Fp ‐resistant and ‐susceptible rainbow trout genetic lines revealed correlated expression of OMV‐packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill‐W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV‐treated cells, similar to the Fp ‐resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV‐treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to comprehensively review the multifaceted factors underlying the successful colonization and infection process of Helicobacter pylori (H. pylori), a prominent Gram-negative pathogen in humans. The focus is on elucidating the functions, mechanisms, genetic regulation, and potential cross-interactions of these elements. Methods: Employing a literature review approach, this study examines the intricate interactions between H. pylori and its host. It delves into virulence factors like VacA, CagA, DupA, Urease, along with phase variable genes, such as babA, babC, hopZ, etc., giving insights about the bacterial perspective of the infection The association of these factors with the infection has also been added in the form of statistical data via Funnel and Forest plots, citing the potential of the virulence and also adding an aspect of geographical biasness to the virulence factors. The biochemical characteristics and clinical relevance of these factors and their effects on host cells are individually examined, both comprehensively and statistically. Results: H. pylori is a Gram-negative, spiral bacterium that successfully colonises the stomach of more than half of the world's population, causing peptic ulcers, gastric cancer, MALT lymphoma, and other gastro-duodenal disorders. The clinical outcomes of H. pylori infection are influenced by a complex interplay between virulence factors and phase variable genes produced by the infecting strain and the host genetic background. A meta-analysis of the prevalence of all the major virulence factors has also been appended. Conclusion: This study illuminates the diverse elements contributing to H. pylori's colonization and infection. The interplay between virulence factors, phase variable genes, and host genetics determines the outcome of the infection. Despite biochemical insights into many factors, their comprehensive regulation remains an understudied area. By offering a panoramic view of these factors and their functions, this study enhances understanding of the bacterium's perspective, i.e. H. pylori's journey from infiltration to successful establishment within the host's stomach. [ABSTRACT FROM AUTHOR]

Abstract Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8+ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8+ T cell responses and increasing the number of CD8+ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. Graphical Abstract

ABSTRACT: Riemerella anatipestifer, belonging to Weeksellaceae family Riemerella, is a bacterium that can infect ducks, geese, and turkeys, causing diseases known as duck infectious serositis, new duck disease, and duck septicemia. We collected diseased materials from ducks on a duck farm in China and then isolated and purified a strain of serotype 1 R. anatipestifer named SX-1. Animal experiments showed that SX-1 is a highly virulent strain with an LD50 value of 101 CFU/mL. The complete genome sequence was obtained. The complete genome sequence of R. anatipestifer SX-1 was 2,112,539 bp; 847 genes were involved in catalytic activity, and 445 genes were related to the cell membrane. The total length of the repetitive sequences was 8746 bp. Four CRISPR loci were predicted in R. anatipestifer strain SX-1, and 4 genomic islands were predicted. Concentration and ultra-high-speed centrifugation were used to extract the outer membrane vesicles of R. anatipestifer SX-1. The OMVs were extracted successfully. Particle size analysis revealed the size and abundance of particles: 147.4 nm, 94.9%; 293.6 nm, 1.1%; 327.2 nm, 1.1%; 397.2 nm, 0.3%; and 371.8 nm, 1.1%. The average size was 173.5 nm. Label-free proteomic technology was used to identify proteins in the outer membrane vesicles. ATCC 11845 served as the reference genome sequence, and 148 proteins were identified using proteomic analysis, which were classified into 5 categories based on their sources. Among them, 24 originated from cytoplasmic proteins, 4 from extracellular secreted proteins, 27 from outer membrane proteins, 10 from periplasmic proteins, and 83 from unknown sources. This study conducted a proteomic analysis of OMVs to provide a theoretical basis for the development of R. anatipestifer OMVs vaccines and adjuvants and lays the foundation for further research on the relationship between the pathogenicity of R. anatipestifer and OMVs.

Introduction: Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. Methods: To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, logand stationary-phase). bMVs from three time points (8  h, 54  h, and 168  h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC–MS/MS). Furthermore, we examined proinflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Results: Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions. Conclusion: Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction. [ABSTRACT FROM AUTHOR]

Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8+ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8+ T cell responses and increasing the number of CD8+ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are membrane-bound structures released by cells and have become significant players in immune system functioning, primarily by facilitating cell-to-cell communication. Immune cells like neutrophils and dendritic cells release EVs containing bioactive molecules that modulate chemotaxis, activate immune cells, and induce inflammation. EVs also contribute to antigen presentation, lymphocyte activation, and immune tolerance. Moreover, EVs play pivotal roles in antimicrobial host defense. They deliver microbial antigens to antigen-presenting cells (APCs), triggering immune responses, or act as decoys to neutralize virulence factors and toxins. This review discusses host and microbial EVs' multifaceted roles in innate and adaptive immunity, highlighting their involvement in immune cell development, antigen presentation, and antimicrobial responses. [ABSTRACT FROM AUTHOR]

Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause acute infections as well as chronic ones in humans. The expression of algD and PpyR genes involved in biofilm formation in clinical isolates of P. aeruginosa in the presence of Escherichia coli Nissle1917 outer membranes vesicles (EcN OMVs) was evaluated. All isolates were tested for biofilm formation. qPCR and disk diffusion were used to identify the expression of algD and PpyR genes, and antimicrobial resistance, respectively. EcN OMVs caused a more significant loss of algD and PpyR expression, compared with the control group. EcN OMVs contain a variety of biomolecules that are capable of influencing the biofilm formation genes. EcN OMVs treatment reduced P. aeruginosa biofilm formation significantly, which emphasizes their positive role in inhibiting biofilm formation. As a result, EcN OMVs can be used as new therapeutic strategies for inhibiting P. aeruginosa biofilm formation.

ABSTRACTCytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed “surface shearing” experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells.IMPORTANCECampylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

Porphyromonas gingivalis uses a variety of mechanisms to actively interact with and promote the hydrolysis of red blood cells (RBCs) to obtain iron in the form of heme. In this study, we investigated the function of lipoprotein PG1881 which was previously shown to be up-regulated during subsurface growth and selectively enriched on outer membrane vesicles (OMVs). Our results show that wildtype strain W83 formed large aggregates encompassing RBCs whereas the PG1881 deletion mutant remained predominately as individual cells. Using a PG1881 antibody, immunofluorescence revealed that the wildtype strain's aggregation to RBCs involves an extracellular matrix enriched with PG1881. Our findings discover that RBCs elicit cell aggregation and matrix formation by P. gingivalis and that this process is promoted by an OMV-specific lipoprotein. We propose this strategy is advantageous for nutrient acquisition as well as dissemination from the oral cavity and survival of this periodontal pathogen.

In the evolving landscape of sustainable biopharmaceutical process development, the utilization of bacteria in the production of various compounds via fermentation has attracted extensive attention from scientists. A successful fermentation process and the release of its associated products hinge on the synergy between an efficient bacterial strain and the formulation of a suitable growth medium. Balancing all nutrient levels of a growth medium to maximize microbial growth and the product quality is quite an intricate task. In this context, significant advancements have been achieved via the strategic implementation of design-of-experiment (DOE) methodologies and the utilization of parallel microbioreactor systems. This work presents a case study of the fermentation growth medium optimization of a Gram-negative bacterium of the Neisseriaceae family that releases outer membrane vesicles (OMVs), which represent a potential vaccine platform. To achieve this, the ability of Sartorius MODDE®13 DOE software to explore multiple variables and their interactions was combined with the functionality of a Sartorius Ambr® 15F parallel microbioreactor system. The findings reported in this study have led to the design of a well-suited fermentation medium for a Gram-negative bacterium and an improvement in the quality of the OMVs produced from it. [ABSTRACT FROM AUTHOR]

Abstract Background Helicobacter pylori (Hp) infects the stomach of 50% of the world’s population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. Methods Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), βIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. Results Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVβ3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. Conclusions OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause acute infections as well as chronic ones in humans. The expression of algD and PpyR genes involved in biofilm formation in clinical isolates of P. aeruginosa in the presence of Escherichia coli Nissle1917 outer membranes vesicles (EcN OMVs) was evaluated. All isolates were tested for biofilm formation. qPCR and disk diffusion were used to identify the expression of algD and PpyR genes, and antimicrobial resistance, respectively. EcN OMVs caused a more significant loss of algD and PpyR expression, compared with the control group. EcN OMVs contain a variety of biomolecules that are capable of influencing the biofilm formation genes. EcN OMVs treatment reduced P. aeruginosa biofilm formation significantly, which emphasizes their positive role in inhibiting biofilm formation. As a result, EcN OMVs can be used as new therapeutic strategies for inhibiting P. aeruginosa biofilm formation. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAMs) promote tumor development and metastasis and are categorized into M1-like macrophages, suppressing tumor cells, and M2-like macrophages. M2-like macrophages, occupying a major role in TAMs, can be repolarized into anti-tumoral phenotypes. In this study, outer membrane vesicles (OMVs) secreted by Escherichia coli Nissle 1917 carry perhexiline (OMV@Perhx) to explore the influence of OMVs and perhexiline on TAM repolarization. OMV@Perhx was internalized by macrophages and regulated the phenotype of TAMs from M2-like to M1-like efficiently to increase the level of tumor suppressor accordingly. Re-polarized macrophages promoted apoptosis and inhibited the mobility of tumor, cells including invasion and migration. The results indicate that OMVs improve the efficacy of perhexiline and also represent a promising natural immunomodulator. Combining OMVs with perhexiline treatments shows powerfully synergistic anti-tumor effects through co-culturing with re-polarized macrophages. This work is promising to exploit the extensive applications of OMVs and chemical drugs, therefore developing a meaningful drug carrier and immunomodulator as well as expanding the purposes of traditional chemical drugs. [ABSTRACT FROM AUTHOR]

Despite the importance of outer membrane vesicles (OMVs) in benthic animal settlement, the underlying molecular mechanism remains elusive. Here, the impact of OMVs and OMVs synthesis-related tolB gene in Mytilus coruscus plantigrade settlement was tested. The OMVs were extracted from Pseudoalteromonas marina through density gradient centrifugation, and a tolB knockout strain, achieved by homologous recombination, was utilized for the investigation. Our results demonstrated that OMVs could significantly enhance M. coruscus plantigrades settlement. Deleting the tolB resulted in downregulation of c-di-GMP, accompanied by a reduction of OMV production, a decline in bacterial motility and increasing biofilm-forming ability. Enzyme treatment resulted in a 61.11% reduction in OMV-inducing activity and a 94.87% reduction in LPS content. Thus, OMVs regulate mussel settlement via LPS, and c-di-GMP is responsible for the OMV-inducing capacity. These findings provide new insights into the interactions between bacteria and mussels. [ABSTRACT FROM AUTHOR]

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen. [ABSTRACT FROM AUTHOR]

Background: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. Methods: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), βIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. Results: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVβ3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. Conclusions: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells. [ABSTRACT FROM AUTHOR]

Gram‐negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram‐negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA‐seq analysis on OMV‐ and mock‐treated plants at 2, 6 and 24 h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune‐related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid‐insensitive 1–associated kinase (BAK1) co‐receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria serve as transporters for the delivery of cargo such as virulence and antibiotic resistance factors. OMVs play a key role in the defense against membrane-targeting antibiotics such as the polymyxin B. Herein, we conducted comparative proteomics of OMVs from paired Klebsiella pneumoniae ATCC 700721 polymyxin-susceptible (polymyxin B MIC = 0.5 mg/L) and an extremely resistant (polymyxin B MIC ≥128 mg/L), following exposure to 2 mg/L of polymyxin B. Comparative profiling of the OMV subproteome of each strain revealed proteins from multiple perturbed pathways, particularly in the polymyxin-susceptible strain, including outer membrane assembly (lipopolysaccharide, O-antigen, and peptidoglycan biosynthesis), cationic antimicrobial peptide resistance, β-lactam resistance, and quorum sensing. In the polymyxin-susceptible strain, polymyxin B treatment reduced the expression of OMV proteins in the pathways related to adhesion, virulence, and the cell envelope stress responses, whereas, in the polymyxin-resistant strain, the proteins involved in LPS biosynthesis, RNA degradation, and nucleotide excision repair were significantly overexpressed in response to polymyxin B treatment. Intriguingly, the key polymyxin resistance enzymes 4-amino-4-deoxy-l-arabinose transferase and the PhoPQ two-component protein kinase were significantly downregulated in the OMVs of the polymyxin-susceptible strain. Additionally, a significant reduction in class A β-lactamase proteins was observed following polymyxin B treatment in the OMVs of both strains, particularly the OMVs of the polymyxin-susceptible strain. These findings shed new light on the OMV subproteome of extremely polymyxin resistant K. pneumoniae, which putatively may serve as active decoys to make the outer membrane more impervious to polymyxin attack. IMPORTANCE OMVs can help bacteria to fight antibiotics not only by spreading antibiotic resistance genes but also by acting as protective armor against antibiotics. By employing proteomics, we found that OMVs have a potential role in shielding K. pneumoniae and acting as decoys to polymyxin attack, through declining the export of proteins (e.g., 4-amino-4-deoxy-l-arabinose transferase) involved in polymyxin resistance. Furthermore, polymyxin B treatment of both strains leads to shedding of the OMVs with perturbed proteins involved in outer membrane remodeling (e.g., LPS biosynthesis) as well as pathogenic potential of K. pneumoniae (e.g., quorum sensing). The problematic extended spectrum beta-lactamases SHV and TEM were significantly reduced in both strains, suggesting that polymyxin B may act as a potentiator to sensitize the bacterium to β-lactam antibiotics. This study highlights the importance of OMVs as “molecular mules” for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxins.

In the evolving landscape of sustainable biopharmaceutical process development, the utilization of bacteria in the production of various compounds via fermentation has attracted extensive attention from scientists. A successful fermentation process and the release of its associated products hinge on the synergy between an efficient bacterial strain and the formulation of a suitable growth medium. Balancing all nutrient levels of a growth medium to maximize microbial growth and the product quality is quite an intricate task. In this context, significant advancements have been achieved via the strategic implementation of design-of-experiment (DOE) methodologies and the utilization of parallel microbioreactor systems. This work presents a case study of the fermentation growth medium optimization of a Gram-negative bacterium of the Neisseriaceae family that releases outer membrane vesicles (OMVs), which represent a potential vaccine platform. To achieve this, the ability of Sartorius MODDE®13 DOE software to explore multiple variables and their interactions was combined with the functionality of a Sartorius Ambr® 15F parallel microbioreactor system. The findings reported in this study have led to the design of a well-suited fermentation medium for a Gram-negative bacterium and an improvement in the quality of the OMVs produced from it.

Alginate is an important polysaccharide in the ocean that supports the growth of marine microorganisms. Many widespread Vibrio species possess alginate lyases and can utilize alginate as a carbon source, but the detailed alginate degradation mechanism in Vibrio remains to be further explored. In this study, we obtained a highly efficient alginate-degrading strain, Vibrio pelagius WXL662, with 11 alginate lyases (VpAly-I to -XI) and further elucidated its molecular mechanism of alginate degradation. Three alginate utilization loci (AUL) were identified in different parts of WXL662's genome, comprising six alginate lyases (VpAly-I, -II, -VIII, -IX, -X, and -XI) and other genes related to alginate degradation. Most of the alginate-degrading genes are strongly induced when alginate is provided as the sole carbon source. Ten alginate lyases (VpAly-I to -X) had been purified and characterized, including six from polysaccharide lyase family 7 (PL7), three from PL17, and one from PL6. These recombinant alginate lyases existing in different cellular locations were active at a wide temperature (10 to 50°C) and pH (4.0 to 9.0) range, with different substrate preferences and diverse degradation products, enabling WXL662 to efficiently utilize alginate in a changing marine environment. Importantly, outer membrane vesicles (OMVs) can act as vectors for alginate lyases (VpAly-II, -V, and -VI) in WXL662. Further investigations of public Vibrio genomes revealed that most alginate-degrading vibrios possess one AUL instead of previously reported "scattered" system. These results emphasize the specific alginate degradation strategy in Vibrio pelagius WXL662, which can be used as a model strain to study the ecological importance of effective alginate-degrading vibrios in the ocean. [ABSTRACT FROM AUTHOR]

ABSTRACT Porphyromonas gingivalis is a keystone oral pathogen that successfully manipulates the human innate immune defenses, resulting in a chronic proinflammatory state of periodontal tissues and beyond. Here, we demonstrate that secreted outer membrane vesicles (OMVs) are deployed by P. gingivalis to selectively coat and activate human neutrophils, thereby provoking degranulation without neutrophil killing. Secreted granule components with antibacterial activity, especially LL-37 and myeloperoxidase (MPO), are subsequently degraded by potent OMV-bound proteases known as gingipains, thereby ensuring bacterial survival. In contrast to neutrophils, the P. gingivalis OMVs are efficiently internalized by macrophages and epithelial cells. Importantly, we show that neutrophil coating is a conserved feature displayed by OMVs of at least one other oral pathogen, namely, Aggregatibacter actinomycetemcomitans. We conclude that P. gingivalis deploys its OMVs for a neutrophil-deceptive strategy to create a favorable inflammatory niche and escape killing. IMPORTANCE Severe periodontitis is a dysbiotic inflammatory disease that affects about 15% of the adult population, making it one of the most prevalent diseases worldwide. Importantly, periodontitis has been associated with the development of nonoral diseases, such as rheumatoid arthritis, pancreatic cancer, and Alzheimer’s disease. Periodontal pathogens implicated in periodontitis can survive in the oral cavity only by avoiding the insults of neutrophils while at the same time promoting an inflamed environment where they successfully thrive. Our present findings show that outer membrane vesicles secreted by the keystone pathogen Porphyromonas gingivalis provide an effective delivery tool of virulence factors that protect the bacterium from being killed while simultaneously activating human neutrophils.

In the last decade, nanoparticle‐based therapeutic modalities have emerged as promising treatment options for cancer and infectious diseases. To improve prognosis, chemotherapeutic and antimicrobial drugs must be delivered selectively to the target sites. Researchers have increasingly focused their efforts on improving drug delivery, with a particular emphasis on cancer and infectious diseases. When drugs are administered systemically, they become diluted and can diffuse to all tissues but only until the immune system intervenes and quickly removes them from circulation. To enhance and prolong the systemic circulation of drugs, nanocarriers have been explored and used; however, nanocarriers have a major drawback in that they can trigger immune responses. Numerous nanocarriers for optimal drug delivery have been developed using innovative and effective biointerface technologies. Autologous cell‐derived drug carriers, such as outer membrane vesicles (OMVs), have demonstrated improved bioavailability and reduced toxicity. Thus, this study investigates the use of biomimetic OMVs as biomimetic vaccine carriers against infections and cancers to improve our understanding in the field of nanotechnology. In addition, discussion on the advantages, disadvantages, and future prospects of OMVs will also be explored. This article is categorized under:Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic DiseaseTherapeutic Approaches and Drug Discovery > Nanomedicine for Infectious DiseaseBiology‐Inspired Nanomaterials > Protein and Virus‐Based Structures [ABSTRACT FROM AUTHOR]

ABSTRACT Outer membrane vesicles (OMVs) of Acinetobacter baumannii DS002 carry proteins which perform selective biological functions. The proteins involved in cell wall/membrane biogenesis and inorganic ion transport and metabolism occupied a significant portion of the 302 proteins associated with OMVs. Interestingly, the TonB-dependent transporters (TonRs), linked to the active transport of nutrients across the energy-deprived outer membrane, are predominant among proteins involved in inorganic ion transport and metabolism. The OMVs of DS002 contain TonRs capable of transporting iron complexed to catecholate, hydroximate, and mixed types of siderophores. Consistent with this observation, the OMVs were firmly bound to ferric-enterobactin (55Fe-Ent) and successfully transported iron into A. baumannii DS002 cells grown under iron-limiting conditions. In addition to the TonRs, OMVs also carry proteins known to promote pathogenesis, immune evasion, and biofilm formation. Our findings provide conclusive evidence for the role of OMVs in the transport of nutrients such as iron and show the presence of proteins with proven roles in pathogenicity and immune response. IMPORTANCE TonB-dependent transporters (TonRs) play a crucial role in transporting nutrients such as iron, nickel, copper, and complex carbohydrates across the energy-deprived outer membrane. Due to their unique structural features, TonRs capture nutrients in an energy-independent manner and transport them across the outer membrane by harvesting energy derived from the inner membrane-localized Ton-complex. In this study, we report the presence of TonRs capable of transporting various nutrients in OMVs and demonstrate their role in capturing and transporting ferric iron complexed with enterobactin into A. baumannii DS002 cells. The OMV-associated TonRs appear to play a critical role in the survival of A. baumannii, listed as a priority pathogen, under nutrient-deprived conditions.

Salmonella is a common pathogen which can secrete outer membrane vesicles (OMVs). However, the effect of OMVs from Salmonella enterica Serovar Typhimurium (S. Typhimurium) of poultry origin on cells of the chicken innate immune system is not well known. In this study, S. Typhimurium OMVs were first isolated from three different poultry strains of Salmonella, Salmonella CVCC542, SALA, and SALB. In order to investigate the effect of OMVs on the maturation of monocytes into macrophages, both bone marrow-derived (BMD) monocytes and macrophage cell line HD11 cells were used. OMVs promoted the formation of monocyte dendrites in both types of cells, enabled BMD cells to become larger, and stimulated expression of LPS-induced TNF-αfactor (LITAF), IL-6, and inducible nitric oxide synthase (iNOS) genes in HD11 cells. These results demonstrated the capability of OMVs to promote the development of chicken monocytes into macrophages and the maturation of macrophages. In order to study the effect of OMVs on the phagocytosis of macrophages, chicken spleen-derived monocytes and HD11 cells were used. Phagocytosis of FITC-Salmonella and FITC-dextran by these two types of cells was enhanced after stimulation with OMVs. To determine which components in OMVs were responsible for the above observed results, OMVs were treated with proteinase K(PK) or polymyxin B (PMB). Both treatments reduced the phagocytosis of FITC-Salmonella by HD11 cells and chicken spleen mononuclear cells and reduced the secretion of IL-1β, LITAF, and IL-6 cytokines. These results demonstrated that Salmonella OMVs activated chicken macrophages and spleen mononuclear cells and the activation was achieved mainly through lipopolysaccharides and membrane proteins. [ABSTRACT FROM AUTHOR]

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

Emergence of antimicrobial-resistant bacteria (AMR) is one of the health major problems worldwide. The scientists are looking for a novel method to treat infectious diseases. Phage therapy is considered a suitable approach for treating infectious diseases. However, there are different challenges in this way. Some biological aspects can probably influence on therapeutic results and further investigations are necessary to reach a successful phage therapy. Bacteriophage activity can influence by bacterial defense system. Bacterial extracellular vesicles (BEVs) are one of the bacterial defense mechanisms which can modify the results of bacteriophage activity. BEVs have the significant roles in the gene transferring, invasion, escape, and spreading of bacteriophages. In this review, the defense mechanisms of bacteria against bacteriophages, especially BEVs secretion, the hidden linkage of BEVs and bacteriophages, and its possible consequences on the bacteriophage activity as well phage therapy will be discussed. [Display omitted] • Emergence of antimicrobial- resistant bacteria is one of the health global problems. • Bacteriophage therapy can be a promising treatment to control infectious diseases caused by antibiotic-resistant bacteria. • Bactetial extracellular vesicles secretion can influence the activity and infectivity of bacteriophages. • Bactetial extracellular vesicles have the significant roles in the gene transferring, invasion, escape and spreading of bacteriophages. [ABSTRACT FROM AUTHOR]

The appearance of multi-resistant strains has contributed to reintroducing polymyxin as the last-line therapy. Although polymyxin resistance is based on bacterial envelope changes, other resistance mechanisms are being reported. Outer membrane vesicles (OMVs) are nanosized proteoliposomes secreted from the outer membrane of Gram-negative bacteria. In some bacteria, OMVs have shown to provide resistance to diverse antimicrobial agents either by sequestering and/or expelling the harmful agent from the bacterial envelope. Nevertheless, the participation of OMVs in polymyxin resistance has not yet been explored in S. Typhi, and neither OMVs derived from hypervesiculating mutants. In this work, we explored whether OMVs produced by the hypervesiculating strains Salmonella Typhi ΔrfaE (LPS synthesis), ΔtolR (bacterial envelope) and ΔdegS (misfolded proteins and σE activation) exhibit protective properties against polymyxin B. We found that the OMVs extracted from S. Typhi ΔtolR and ΔdegS protect S. Typhi WT from polymyxin B in a concentration-depending manner. By contrast, the protective effect exerted by OMVs from S. Typhi WT and S. Typhi ΔrfaE is much lower. This effect is achieved by the sequestration of polymyxin B, as assessed by the more positive Zeta potential of OMVs with polymyxin B and the diminished antibiotic’s availability when coincubated with OMVs. We also found that S. Typhi ΔtolR exhibited an increased MIC of polymyxin B. Finally, we determined that S. Typhi ΔtolR and S. Typhi ΔdegS, at a lesser level, can functionally and transiently transfer the OMV-mediated polymyxin B resistance to susceptible bacteria in cocultures. This work shows that mutants in genes related to OMVs biogenesis can release vesicles with improved abilities to protect bacteria against membrane-active agents. Since mutations affecting OMV biogenesis can involve the bacterial envelope, mutants with increased resistance to membrane-acting agents that, in turn, produce protective OMVs with a high vesiculation rate (e.g., S. Typhi ΔtolR) can arise. Such mutants can functionally transfer the resistance to surrounding bacteria via OMVs, diminishing the effective concentration of the antimicrobial agent and potentially favoring the selection of spontaneous resistant strains in the environment. This phenomenon might be considered the source for the emergence of polymyxin resistance in an entire bacterial community.

The appearance of multi-resistant strains has contributed to reintroducing polymyxin as the last-line therapy. Although polymyxin resistance is based on bacterial envelope changes, other resistance mechanisms are being reported. Outer membrane vesicles (OMVs) are nanosized proteoliposomes secreted from the outer membrane of Gram-negative bacteria. In some bacteria, OMVs have shown to provide resistance to diverse antimicrobial agents either by sequestering and/or expelling the harmful agent from the bacterial envelope. Nevertheless, the participation of OMVs in polymyxin resistance has not yet been explored in S. Typhi, and neither OMVs derived from hypervesiculating mutants. In this work, we explored whether OMVs produced by the hypervesiculating strains Salmonella Typhi Δ rfaE (LPS synthesis), Δ tolR (bacterial envelope) and Δ degS (misfolded proteins and σ E activation) exhibit protective properties against polymyxin B. We found that the OMVs extracted from S. Typhi Δ tolR and Δ degS protect S. Typhi WT from polymyxin B in a concentration-depending manner. By contrast, the protective effect exerted by OMVs from S. Typhi WT and S. Typhi Δ rfaE is much lower. This effect is achieved by the sequestration of polymyxin B, as assessed by the more positive Zeta potential of OMVs with polymyxin B and the diminished antibiotic's availability when coincubated with OMVs. We also found that S. Typhi Δ tolR exhibited an increased MIC of polymyxin B. Finally, we determined that S. Typhi Δ tolR and S. Typhi Δ degS , at a lesser level, can functionally and transiently transfer the OMV-mediated polymyxin B resistance to susceptible bacteria in cocultures. This work shows that mutants in genes related to OMVs biogenesis can release vesicles with improved abilities to protect bacteria against membrane-active agents. Since mutations affecting OMV biogenesis can involve the bacterial envelope, mutants with increased resistance to membrane-acting agents that, in turn, produce protective OMVs with a high vesiculation rate (e.g., S. Typhi Δ tolR) can arise. Such mutants can functionally transfer the resistance to surrounding bacteria via OMVs, diminishing the effective concentration of the antimicrobial agent and potentially favoring the selection of spontaneous resistant strains in the environment. This phenomenon might be considered the source for the emergence of polymyxin resistance in an entire bacterial community. [ABSTRACT FROM AUTHOR]

Teredinibacter turnerae is a cultivable cellulolytic Gammaproteobacterium (Cellvibrionaceae) that commonly occurs as an intracellular endosymbiont in the gills of wood-eating bivalves of the family Teredinidae (shipworms). The genome of T. turnerae encodes a broad range of enzymes that deconstruct cellulose, hemicellulose, and pectin and contribute to wood (lignocellulose) digestion in the shipworm gut. However, the mechanisms by which T. turnerae secretes lignocellulolytic enzymes are incompletely understood. Here, we show that T. turnerae cultures grown on carboxymethyl cellulose (CMC) produce membrane vesicles (MVs) that include a variety of proteins identified by LC-MS/MS as carbohydrate-active enzymes (CAZymes) with predicted activities against cellulose, hemicellulose, and pectin. Reducing sugar assays and zymography confirm that these MVs exhibit cellulolytic activity, as evidenced by the hydrolysis of CMC. Additionally, these MVs were enriched with TonB -dependent receptors, which are essential to carbohydrate and iron acquisition by free-living bacteria. These observations indicate a potential role for MVs in lignocellulose utilization by T. turnerae in the free-living state, suggest possible mechanisms for host-symbiont interaction, and may be informative for commercial applications such as enzyme production and lignocellulosic biomass conversion., Competing Interests: Conflict of interest statement The authors report no conflict of interest. The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.

Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destruction. However, how pathogens respond to irradiation stress has not yet been fully elucidated. Here, we observed that Pseudomonas aeruginosa PAO1 could release nucleic acids into the extracellular environment under X-ray irradiation. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), X-ray irradiation was observed to induce outer membrane vesicle (OMV) formation in P. aeruginosa PAO1. The size distribution of the OMVs of the irradiated PAO1 was similar to that of the OMVs of the non-irradiated PAO1 according to nanoparticle tracking analysis (NTA). The pyocin-related proteins are involved in OMV production in P. aeruginosa PAO1 under X-ray irradiation conditions, and that this is regulated by the key SOS gene recA. The OMV production was significantly impaired in the irradiated PAO1 Δlys mutant, suggesting that Lys endolysin is associated with OMV production in P. aeruginosa PAO1 upon irradiation stress. Meanwhile, no significant difference in OMV production was observed between PAO1 lacking the pqsR, lasR, or rhlR genes and the parent strain, demonstrating that the irradiation-induced OMV biosynthesis of P. aeruginosa was independent of the Pseudomonas quinolone signal (PQS).

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.