Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that contain biologically active proteins and perform diverse biological processes. Unlike other secretion mechanisms, OMVs enable bacteria to secrete insoluble molecules in addition to and in complex with soluble material. OMVs allow enzymes to reach distant targets in a concentrated, protected, and targeted form. OMVs also play roles in bacterial survival: Their production is a bacterial stress response and important for nutrient acquisition, biofilm development, and pathogenesis. Key characteristics of OMV biogenesis include outward bulging of areas lacking membrane-peptidoglycan bonds, the capacity to upregulate vesicle production without also losing outer membrane integrity, enrichment or exclusion of certain proteins and lipids, and membrane fission without direct energy from ATP/GTP hydrolysis. Comparisons of similar budding mechanisms from diverse biological domains have provided new insight into evaluating mechanisms for outer membrane vesiculation.

Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.In nearly every case, virulence factors of Gram-negative pathogens are secreted products that enhance the survival of the bacteria and/or damage the host. Secretion of virulence factors by Gram-negative pathogens is complicated by the fact that the bacterial envelope consists of two lipid bilayers, the inner and outer membrane, and the periplasm in between. Gram-negative pathogens have developed many strategies, some specific to pathogens, to enable active virulence factors to gain access to the extracellular environment, typically the tissues or bloodstream of the host organism (Henderson et al. 2004). The Type II and Type V secretion systems are two-step processes in which proteins are transported first through the inner membrane (IM) and then through the outer membrane (OM). For secretion via the Type I, Type III, and Type IV secretion systems, the material is transferred directly into the extracellular milieu or into another cell. The Type III system is specific for the transport of factors by pathogenic bacteria. All of these secretion systems secrete individual proteins or small complexes. This review examines secretion via OM vesicles, a distinct "Type VI" mechanism that enables bacteria to secrete a large, complex group of proteins and lipids into the extracellular milieu.Both pathogenic and nonpathogenic species of Gram- Vesicles are a means by which bacteria interact with prokaryotic and eukaryotic cells in their environment. Some of the best-characterized vesicles are those produced by pathogens. Biochemical analysis and functional characterization of pathogen-derived outer membrane vesicles demonstrate that this secretory pathway has been usurped by pathogens for the transport of active virulence factors to host cells (Table 1). Naturally produced OM vesicles from pathogenic bacteria contain adhesins, toxins, and immunomodulatory compounds, and they directly mediate bacterial binding and invasion, cause cytotoxicity, and modulate the host immune response. By participating in such diverse aspects of the host-pathogen interaction, OM vesicles are potent bacterial virulence factors. Formation of bacterial OM vesiclesNaturally produced bacterial vesicles are discrete, closed OM blebs produced by growing cells, not products of cell lysis or cell death (Mug-Opstelte...

SUMMARY Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.

SummaryConditions that impair protein folding in the Gramnegative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

BackgroundOuter membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection.ResultsWe found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs.ConclusionsThese data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth. Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells. Enterotoxigenic E. coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation. Vesicle protein profiles were similar but not identical to outer membranes and differed between strains. Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal. Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles. Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles. In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles. LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions. Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles. The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains. Enterotoxigenic Escherichia coli (ETEC)1 is an important pathogen responsible for traveler's diarrhea and causes more than 700,000 childhood deaths due to diarrhea per year in third-world countries (1-4). ETEC produce several toxins, including the heat labile enterotoxin (LT), which disrupts electrolyte balance in the gut endothelium (2,5,6). LT is an AB 5 toxin that binds Gal␤1,3GalNAc␤1(NeuAc␣2,3),4Gal␤1,4Glc ceramide (G M1 ) ganglioside on epithelial cells via its B subunit (7). Once internalized by the epithelial cell, the enzymatic A subunit catalyzes the ADP-ribosylation of the G s␣ subunit in the adenylate cyclase pathway leading to an increase in cAMP (2, 8 -10). Elevated cAMP levels cause chloride efflux and, thereby, diarrhea. Despite intimate knowledge of its structure and function (1, 11), the mode of LT secretion from ETEC remains unclear.LT shares more than 80% sequence homology with another AB 5 toxin, Vibrio cholerae toxin, CT (2). Purified CT and LT exhibit equivalent activity in bioassays; however, disease caused by V. cholerae is more severe than that caused by ETEC (1). This suggests V. cholerae-and ETEC-mediated toxicity may be partially dependent upon the efficiency of toxin secretion (2). The signal sequences of the A and B subunits from both LT and CT are cleaved upon entrance into the periplasmic space after transport across the cytoplasmic membrane (11-13). The similarities stop in the periplasm, however; soluble CT is secreted from the cell, whereas soluble LT is reported to remain in the periplasm (3, 6). E. coli transformed with a CT-expressing plasmid does not efficiently secrete CT, whereas V. cholerae will secrete LT e...

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over-or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the E cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.Release of outer membrane (OM) vesicles has been observed for all gram-negative bacteria studied to date (reviewed in references 5, 15, and 16). Native vesicles are rounded structures with lumenal periplasmic components bounded by an outer layer of outer membrane proteins (Omps) and lipids (16). For Escherichia coli, our laboratory has reported that strain DH5␣ releases 0.23% of Omps F and C and 0.14% of OmpA into vesicles (12); other investigators have found vesicles to account for 0.2 to 0.5% of bacterial culture material (9,20). Electron microscopy studies reveal bulging of the OM and subsequent fission of vesicles containing electron-dense material (16). These biochemical and microscopic observations suggest that OM vesicles are formed from protrusions that are pinched off from the OM in a manner that leads to the inclusion of periplasmic material.The wide variety of strains and diversity of environments for which vesiculation has been observed suggest an important role for vesicle production in gram-negative bacterial growth and survival (5,15,16). Vesicle production varies with growth phase and nutrient availability, and vesicle-associated enzymes may aid in nutrient scavenging. Vesicle-mediated transfer of toxic components to other bacteria can eliminate competing species. In addition, interactions between eukaryotic cells and vesicles from pathogenic bacteria suggest a role for vesicles in pathogenesis (13).We have conducted a screen to generate and identify mutants in E. coli that exhibit altered vesiculation levels. Both vesicle-overproducing and -underproducing mutants are of interest, ...