Production of extracellular membrane vesicles plays an important role in communication in bacterial populations and in bacteria-host interactions. Vesicles as carriers of various regulatory and signaling molecules may be potentially used as disease biomarkers and promising therapeutic agents, including vaccine preparations. The composition of membrane vesicles has been deciphered for a limited number of Gram-negative and Gram-positive bacteria. In this work, for the first time, extracellular membrane vesicles of a streptomycin-resistant strain Bacillus pumilus 3-19, a producer of extracellular guanyl-preferring ribonuclease binase, are isolated, visualized, and characterized by their genome and proteome composition. It has been established that there is no genetic material in the vesicles and the spectrum of the proteins differs depending on the phosphate content in the culture medium of the strain. Vesicles from a phosphate-deficient medium carry 49 unique proteins in comparison with 101 from a medium with the high phosphate content. The two types of vesicles had 140 mutual proteins. Flagellar proteins, RNase J, which is the main enzyme of RNA degradosomes, phosphatases, peptidases, iron transporters, signal peptides, were identified in vesicles. Antibiotic resistance proteins and amyloid-like proteins whose genes are present in B. pumilus 3-19 cells are absent. Phosphate deficiency-induced binase was found only in vesicles from a phosphate-deficient medium.

The aim of this study was to identify a Bifidobacterium strain that improves the performance of Limosilactobacillus reuteri DSM 17938. Initial tests showed that Bifidobacterium longum subsp. longum strains boosted the growth of DSM 17938 during in vivo-like conditions. Further characterization revealed that one of the strains, BG-L47, had better bile and acid tolerance compared to BG-L48, as well as mucus adhesion compared to both BG-L48 and the control strain BB536. BG-L47 also had the capacity to metabolize a broad range of carbohydrates and sugar alcohols. Mapping of glycoside hydrolase (GH) genes of BG-L47 and BB536 revealed many GHs associated with plant-fiber utilization. However, BG-L47 had a broader phenotypic fiber utilization capacity. In addition, B. longum subsp. longum cells boosted the bioactivity of extracellular membrane vesicles (MV) produced by L. reuteri DSM 17938 during co-cultivation. Secreted 5\'-nucleotidase (5\'NT), an enzyme that converts AMP into the signal molecule adenosine, was increased in MV boosted by BG-L47. The MV exerted an improved antagonistic effect on the pain receptor transient receptor potential vanilloid 1 (TRPV1) and increased the expression of the immune development markers IL-6 and IL-1ß in a peripheral blood mononuclear cell (PBMC) model. Finally, the safety of BG-L47 was evaluated both by genome safety assessment and in a human safety study. Microbiota analysis showed that the treatment did not induce significant changes in the composition. In conclusion, B. longum subsp. longum BG-L47 has favorable physiological properties, can boost the in vitro activity of L. reuteri DSM 17938, and is safe for consumption, making it a candidate for further evaluation in probiotic studies.
OBJECTIVE: By using probiotics that contain a combination of strains with synergistic properties, the likelihood of achieving beneficial interactions with the host can increase. In this study, we first performed a broad screening of Bifidobacterium longum subsp. longum strains in terms of synergistic potential and physiological properties. We identified a superior strain, BG-L47, with favorable characteristics and potential to boost the activity of the known probiotic strain Limosilactobacillus reuteri DSM 17938. Furthermore, we demonstrated that BG-L47 is safe for consumption in a human randomized clinical study and by performing a genome safety assessment. This work illustrates that bacteria-bacteria interactions differ at the strain level and further provides a strategy for finding and selecting companion strains of probiotics.

Staphylococcus aureus is an important bacterial pathogen that causes a wide variety of human diseases in community and hospital settings. S. aureus employs a diverse array of virulence factors, both surface-associated and secreted, to promote colonization, infection, and immune evasion. Over the past decade, a growing body of research has shown that S. aureus generates extracellular membrane vesicles (MVs) that package a variety of bacterial components, many of which are virulence factors. In this review, we summarize recent advances in our understanding of S. aureus MVs and highlight their biogenesis, cargo, and potential role in the pathogenesis of staphylococcal infections. Lastly, we present some emerging questions in the field.

Membranous Extracellular Vesicles (EVs) of Gram-negative bacteria are a secretion and delivery system that can disseminate bacterial products and interact with hosts and the environment. EVs of nonpathogenic bacteria deliver their contents by endocytosis into eukaryotic cells, however, no evidence exists for a fusion delivery mechanism. Here, we describe the fusion of exposed to space/Mars-like stressors simulated on the International Space Station vesicles (E-EVs) from Komagataeibacter oboediens to different types of model planar membranes in comparison with the EVs of the ground-based reference strain. The most reliable fusion was achieved with PC:PE:ergosterol or sterol-free PC:PE bilayers. The relative permeability ratio (PK+/PCl-) estimated from the shift of zero current potential according to Goldman-Hodgkin-Katz equation consisted of 4.17 ± 0.48, which coincides with preferential cation selectivity of the EV endogenous channels. The increase in membrane potential from 50 mV to 100 mV induced the fusion of E-EVs with all tested lipid compositions. The fusion of model exosomes with planar bilayer lipid membranes was confirmed by separate step-like increases in its conductance. In contrast, the ground-based reference K. oboediens EVs never induced the fusion event. In our study, we show membrane lipidome perturbations and increased protein aggregation occurred in the exposed samples in the harsh environment when outer membranes of K. oboediens acquired the capability of both homo- and heterotypic fusion possibly by altered membrane fluidity and the pore-forming capability.

Extracellular-membrane vesicles (EMVs) are spherical buds of the extracellular membrane, commonly produced by Gram-negative bacteria, known to mediate intricate inter-kingdom communication. In this context, comprehensive research dissecting the role of EMVs in one of the most complex nature-occurring molecular dialogues, rhizobium-legume symbiosis, has been so far neglected. During the different stages of the symbiotic process, rhizobia and their host plants establish a very specific and controlled intercellular trafficking of signal molecules. Thus, as conveyors of a broad range of molecules into the target cell, EMVs are gaining weight in the field. Here, we describe a detailed protocol to isolate EMVs from bacteroids of legume nodules, opening a new door for discovering new authors of the symbiotic process.

Rhizobia are a group of soil proteobacteria that are able to establish a symbiotic interaction with legumes. These bacteria are capable to fix atmospheric nitrogen into ammonia within specific plant root organs called nodules. The rhizobia-legume interaction is established by a complex molecular dialogue that starts with flavonoids exudated by the plant roots. In response, signaling molecules known as Nod factors (NFs) are secreted by the bacteria. These factors are sensed by specific plant receptors that trigger a downstream signaling cascade leading to rhizobium-specific intracellular colonization of the root hair via the formation of infection threads and the eventual development of nodules on roots. In these organs, rhizobia can fix nitrogen from the atmosphere for the plant in exchange for photosynthates and the appropriate environment for nitrogen fixation. Recently, it has been demonstrated that extracellular membrane vesicles (EMVs) produced by some rhizobia carry NFs. EMVs are proteolipidic structures that are secreted to the milieu from the bacterial membranes and are involved in several important biological processes, including intercellular communication. Thus far, little is known about rhizobia vesicles, and further studies are needed to understand their functions, including their role as transporting vessels of signaling molecules during the process of symbiosis. Here, we present a detailed protocol to isolate high-purity EMVs from free-living cultured rhizobia, test their integrity, and quantify their abundance.

OBJECTIVE: Extracellular membrane vesicles (MVs) produced by Staphylococcus aureus in planktonic cultures encapsulate a diverse cargo of bacterial proteins, nucleic acids, and glycopolymers that are protected from destruction by external factors. δ-toxin, a member of the phenol soluble modulin family, was shown to be critical for MV biogenesis. Amyloid fibrils co-purified with MVs generated by virulent, community-acquired S. aureus strains, and fibril formation was dependent on expression of the S. aureus δ-toxin gene (hld). Mass spectrometry data confirmed that the amyloid fibrils were comprised of δ-toxin. Although S. aureus MVs were produced in vivo in a localized murine infection model, amyloid fibrils were not observed in the in vivo setting. Our findings provide critical insights into staphylococcal factors involved in MV biogenesis and amyloid formation.

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5\'-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1β and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

Bacterial cells release nanometer-sized extracellular membrane vesicles (MVs) to deliver cargo molecules for use in mediating various biological processes. However, the detailed processes of transporting these cargos from MVs to recipient cells remain unclear because of the lack of imaging techniques to image nanometer-sized fragile vesicles in a living bacterial cell surface. Herein, we quantitatively demonstrated that the direct binding of MV to the cell surface significantly promotes hydrophobic quorum-sensing signal (C16-HSL) transportation to the recipient cells. Moreover, we analyzed the MV-binding process in the Paracoccus denitrificans cell surface using high-speed atomic force microscopy phase imaging. Although MV shapes were unaltered after binding to the cell surface, the physical properties of a group of single MV particles were shifted. Additionally, the phase shift values of MVs were higher than that of the cell\'s surfaces upon binding, whereas the phase shift values of the group of MVs were decreased during observation. The shifting physical properties occurred irreversibly only once for each MV during the observations. The decreasing phase shift values indicated alterations of chemical components in the MVs as well, thereby suggesting the dynamic process in which single MV particles deliver their hydrophobic cargo into the recipient cell. IMPORTANCE Compared to the increasing knowledge about MV release mechanisms from donor cells, the mechanism by which recipient cells receive cargo from MVs remains unknown. Herein, we have successfully imaged single MV-binding processes in living bacterial cell surfaces. Accordingly, we confirmed the shift in the MV hydrophobic properties after landing on the cell surface. Our results showed the detailed states and the attaching process of a single MV into the cell surface and can aid the development of a new model for MV reception into Gram-negative bacterial cell surfaces. The insight provided by this study is significant for understanding MV-mediated cell-cell communication mechanisms. Moreover, the AFM technique presented for nanometer-scaled mapping of dynamic physical properties alteration on a living cell could be applied for the analyses of various biological phenomena occurring on the cell surface, and it gives us a new view into the understanding of the phenotypes of the bacterial cell surface.

Pneumococcal infections represent a global health threat, which requires novel vaccine developments. Extracellular vesicles are secreted from most cells, including prokaryotes, and harbor virulence factors and antigens. Hence, bacterial membrane vesicles (MVs) may induce a protective immune response. For the first time, we formulate spray-dried gram-positive pneumococcal MVs-loaded vaccine microparticles using lactose/leucine as inert carriers to enhance their stability and delivery for pulmonary immunization. The optimized vaccine microparticles showed a mean particle size of 1-2 µm, corrugated surface, and nanocrystalline nature. Their aerodynamic diameter of 2.34 µm, average percentage emitted dose of 88.8%, and fine powder fraction 79.7%, demonstrated optimal flow properties for deep alveolar delivery using a next-generation impactor. Furthermore, confocal microscopy confirmed the successful encapsulation of pneumococcal MVs within the prepared microparticles. Human macrophage-like THP-1 cells displayed excellent viability, negligible cytotoxicity, and a rapid uptake around 60% of fluorescently labeled MVs after incubation with vaccine microparticles. Moreover, vaccine microparticles increased the release of pro-inflammatory cytokines tumor necrosis factor and interleukin-6 from primary human peripheral blood mononuclear cells. Vaccine microparticles exhibited excellent properties as promising vaccine candidates for pulmonary immunization and are optimal for further animal testing, scale-up and clinical translation.