Special delivery: vesicle trafficking in prokaryotes

Although the observation that Gram-negative bacteria produce outer membrane vesicles (MVs) was made over 40 years ago, their biological roles have become a focus of study only within the past 10 years. Recent progress in this area has revealed that bacterial MVs are utilized for several processes including delivery of toxins to eukaryotic cells, protein and DNA transfer between bacterial cells, and trafficking of cell-cell signals. Some of these roles appear to be generalized among the Gram-negative bacteria while others are restricted to specific bacterial species/strains. Here we review the known roles of MVs, propose other roles for MVs in mediating interspecies and inter-kingdom communication, and discuss the mechanism of MV formation.     

Multifunctional membrane vesicles in Pseudomonas aeruginosa

Gram‐negative bacteria secrete small particles called membrane vesicles (MVs) into the extracellular milieu. While MVs have important roles in delivering toxins from pathogenic bacteria to eukaryotic cells, these vesicles also play ecological roles necessary for survival in various environmental conditions. Pseudomonas aeruginosa, which lives in soil, ocean, plant, animal and human environments, has become a model organism for studying these small extracellular particles. Such studies have increased our understanding of the function and biogenesis of bacterial MVs. Pseudomonas aeruginosa MVs possess versatile components and chemical substances with unique structures. These characteristics allow MVs to play their multifunctional biological roles, including microbial interaction, maintenance of biofilm structure and host infection. This review summarizes the comprehensive biochemical and physiochemical properties of MVs derived from P. aeruginosa. These studies will help us understand their biological roles of MVs not only in pathogenicity but also in microbial ecology. Also, the mechanisms of MV production, as currently understood, are discussed.     

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-β were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.     

Characterization of phospholipids in membrane vesicles derived from Pseudomonas aeruginosa

Many Gram-negative bacteria release membrane vesicles (MVs), but their phospholipid properties are poorly understood. Phosphatidylglycerol was present at high levels in MVs derived from Pseudomonas aeruginosa, but not in the cellular outer membrane. The ratio of stearic acid in MVs was high compared to that in the cellular outer membrane. These findings suggest that membrane rigidity is associated with MV biogenesis.     

Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-alpha, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4(+) T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4(+) T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.     

Biogenesis of bacterial membrane vesicles

Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo . Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein–peptidoglycan (OM–PG) and OM protein–inner membrane protein (OM–PG–IM) interactions within the envelope structure. Modulation of OM–PG and OM–PG–IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM–PG and OM–PG–IM associations within the envelope structure, thus releasing OM as MVs.     

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.     

Microvesicles as promising biological tools for diagnosis and therapy

ABSTRACT

Introduction: Shed by most cells, in response to a myriad of stimuli, extracellular vesicles (EVs) carry proteins, lipids, and various nucleic acids. EVs encompass diverse subpopulations differing for biogenesis and content. Among these, microvesicles (MVs) derived from plasma membrane, are key regulators of physiopathological cellular processes including cancer, inflammation and infection. This review is unique in that it focuses specifically on the MVs as a mediator of information transfer. In fact, few proteomic studies have rigorously distinguished MVs from exosomes.

Areas covered: Aim of this review is to discuss the proteomic analyses of the MVs. Many studies have examined mixed populations containing both exosomes and MVs. We discuss MVs’ role in cell-specific interactions. We also show their emerging roles in therapy and diagnosis.

Expert commentary: We see MVs as therapeutic tools for potential use in precision medicine. They may also have potential for allowing the identification of new biomarkers. MVs represent an invaluable tool for studying the cell of origin, which they closely represent, but it is critical to build a repository with data from MVs to deepen our understanding of their molecular repertoire and biological functions.     

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis,properties, functions and biomimetic models

BackgroundMatrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100–300 nm diameter harboring the biochemical machinery needed to induce mineralization.Scope of the reviewThe review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs.Major conclusionsMVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies.General significanceMVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.     

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics.

Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.     

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.     

Effects of replacing the unreliable cDNA microarray measurements on the disease classification based on gene expression profiles and functional modules

MOTIVATION: Microarrays datasets frequently contain a large number of missing values (MVs), which need to be estimated and replaced for subsequent data mining. The focus of the paper is to study the effects of different MV treatments for cDNA microarray data on disease classification analysis. RESULTS: By analyzing five datasets, we demonstrate that among three kinds of classifiers evaluated in this study, support vector machine (SVM) classifiers are robust to varied MV imputation methods [e.g. replacing MVs by zero, K nearest-neighbor (KNN) imputation algorithm, local least square imputation and Bayesian principal component analysis], while the classification and regression tree classifiers are sensitive in terms of classification accuracy. The KNNclassifiers built on differentially expressed genes (DEGs) are robust to the varied MV treatments, but the performances of the KNN classifiers based on all measured genes can be significantly deteriorated when imputing MVs for genes with larger missing rate (MR) (e.g. MR > 5%). Generally, while replacing MVs by zero performs relatively poor, the other imputation algorithms have little difference in affecting classification performances of the SVM or KNN classifiers. We further demonstrate the power and feasibility of our recently proposed functional expression profile (FEP) approach as means to handle microarray data with MVs. The FEPs, which are derived from the functional modules that are enriched with sets of DEGs and thus can be consistently identified under varied MV treatments, achieve precise disease classification with better biological interpretation. We conclude that the choice of MV treatments should be determined in context of the later approaches used for disease classification. The suggested exclusion criterion of ignoring the genes with larger MR (e.g. >5%), while justifiable for some classifiers such as KNN classifiers, might not be considered as a general rule for all classifiers.     

α-2-Macroglobulin induces the shedding of microvesicles from cutaneous wound myofibroblasts

Microvesicles (MVs) are recognized as an important class of cell-to-cell messengers. Although the properties of MVs are increasingly documented, the mechanisms regulating MV biogenesis remain debated. Myofibroblasts are a key cellular component of wound healing and have been shown to produce MVs upon stimulation with serum. However, the mediator(s) responsible for the observed effect of serum on MV release have yet to be identified. To isolate the molecule(s) of interest, serum proteins were sequentially separated using chromatography, selective precipitation, and electrophoresis. MV production was assessed throughout the purification and after stimulation of myofibroblasts with two potent purified molecules. α-2-Macroglobulin (A2M) was thereby found to dose-dependently stimulate MV release. We confirmed the presence of the A2M receptor, low-density lipoprotein receptor-related protein-1 (LRP1), on myofibroblasts. Inhibition of LRP1 resulted in a significant decrease in MV production. Together, our results suggest that A2M positively regulates MV shedding through the activation of LRP1 on myofibroblasts.     

Membrane Vesicles: A Common Feature in the Extracellular Matter of Cold-Adapted Antarctic Bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22^T, Shewanella vesiculosa M7^T, Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23^T, and Marinobacter guineae M3B^T) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22^T, the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22^T obtained at 4 and 18°C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

Microvesicles Derived from Adult Human Bone Marrow and Tissue Specific Mesenchymal Stem Cells Shuttle Selected Pattern of miRNAs

Background

Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).

Methodology/Principal Findings

MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.

Conclusions

This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.     

Membrane vesicles: an overlooked component of the matrices of biofilms

The matrix helps define the architecture and infrastructure of biofilms and also contributes to their resilient nature. Although many studies continue to define the properties of both gram-positive and gram-negative bacterial biofilms, there is still much to learn, especially about how structural characteristics help bridge the gap between the chemistry and physical aspects of the matrix. Here, we show that membrane vesicles (MVs), structures derived from the outer membrane of gram-negative bacteria, are a common particulate feature of the matrix of Pseudomonas aeruginosa biofilms. Biofilms grown using different model systems and growth conditions were shown to contain MVs when thin sectioned for transmission electron microscopy, and mechanically disrupted biofilms revealed MVs in association with intercellular material. MVs were also isolated from biofilms by employing techniques for matrix isolation and a modified MV isolation protocol. Together these observations verified the presence and frequency of MVs and indicated that MVs were a definite component of the matrix. Characterization of planktonic and biofilm-derived MVs revealed quantitative and qualitative differences between the two and indicated functional roles, such as proteolytic activity and binding of antibiotics. The ubiquity of MVs was supported by observations of biofilms from a variety of natural environments outside the laboratory and established MVs as common biofilm constituents. MVs appear to be important and relatively unacknowledged particulate components of the matrix of gram-negative or mixed bacterial biofilms.     

Vaccinia virus A26 and A27 proteins form a stable complex tethered to mature virions by association with the A17 transmembrane protein

During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.