Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was ~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.     

Interplay between two quorum sensing-regulated pathways,violacein biosynthesis and VacJ/Yrb,dictates outer membrane vesicle biogenesis in Chromobacterium violaceum

Outer membrane vesicles (OMVs) are lipid nanoparticles released by Gram-negative bacteria, which play multiple roles in bacterial physiology and adaptation to diverse environments. In this work, we demonstrate that OMVs released by the environmental pathogen Chromobacterium violaceum deliver the antimicrobial compound violacein to competitor bacteria, mediating its toxicity in vivo at a long distance. OMVs purified by ultracentrifugation from the wild-type strain, but not from a violacein-abrogated mutant ΔvioABCDE, contained violacein and inhibited several Gram-positive bacteria. Competition tests using co-culture and transwell assays indicated that the C. violaceum wild-type strain killed Staphylococcus aureus better than the ΔvioABCDE mutant strain. We found that C. violaceum achieves growth phase-dependent OMV release by the concerted expression of two quorum sensing (QS)-regulated pathways, namely violacein biosynthesis and VacJ/Yrb system. Although both pathways were activated at high cell density in a QS-dependent manner, the effect on vesiculation was the opposite. While the ΔvioABCDE mutant produced twofold fewer vesicles than the wild-type strain, indicating that violacein induces OMV biogenesis for its own delivery, the ΔvacJ and ΔyrbE mutants were hypervesiculating strains. Our findings uncovered QS-regulated pathways involved in OMV biogenesis used by C. violaceum to package violacein into OMVs for interbacterial competition.     

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.     

Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.     

Helicobacter pylori Growth Stage Determines the Size,Protein Composition,and Preferential Cargo Packaging of Outer Membrane Vesicles

Gram‐negative bacteria release outer membrane vesicles (OMVs) as part of their normal growth that contain a range of cargo from their parent bacterium, including DNA, RNA, and proteins. The protein content of OMVs is suggested to be similar in composition to various sub‐cellular locations of their parent bacterium. However, very little is known regarding the effect of bacterial growth stage on the size, content, and selective packaging of proteins into OMVs. In this study, the global proteome of Helicobacter pylori and their OMVs throughout bacterial growth are examined to determine if bacterial growth stage affected OMV cargo composition. Analysis of OMVs produced by H. pylori reveals that bacterial growth stage affects the size, composition, and selection of protein cargo into OMVs. Proteomic analysis identifies that the proteome of H. pylori OMVs is vastly different throughout bacterial growth and that OMVs contain a range of proteins compared to their parent bacteria. In addition, bacterial growth stage affects the ability of OMVs to induce the production of IL‐8 by human epithelial cells. Therefore, the findings identify that the size, proteome, and immunogenicity of OMVs produced during various stages of bacterial growth is not comparable. Collectively, these findings highlight the importance of considering the bacterial growth stage from which OMVs are isolated, as this will impact their size, protein composition, and ultimately their biological functions.     

Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes

The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane‐bound compartment termed Legionella‐containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small‐angle X‐ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co‐incubation experiments showed a dose‐ and time‐dependent binding of fluorophore‐labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4°C. Purified OMVs induced tumour necrosis factor‐α production in human macrophages at concentrations starting at 300 ng ml^?1. Experiments on HEK293‐TLR2 and TLR4/MD‐2 cell lines demonstrated a dominance of TLR2‐dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.     

Outer membrane vesicles as platform vaccine technology

Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications.     

Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate‐active enzymes with versatile polysaccharide‐degrading capacity

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate‐active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non‐cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV‐pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose‐degrading enzyme cocktail by 2.4‐fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro‐slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.     

Insecticidal Activity Associated with the Outer Membrane Vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli

Gram-negative bacteria constitutively secrete native outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress in this area has revealed that OMVs are essential for bacterial survival and pathogenesis, the mechanism of vesicle formation and the biological roles of OMVs have not been clearly defined. Using a proteomics approach, we identified 141 protein components of Escherichia coli-derived native OMVs with high confidence; two separate analyses yielded identifications of 104 and 117 proteins, respectively, with 80 proteins overlapping between the two trials. In the group of identified proteins, the outer membrane proteins were highly enriched, whereas inner membrane proteins were lacking, suggesting that a specific sorting mechanism for vesicular proteins exists. We also identified proteins involved in vesicle formation, the removal of toxic compounds and attacking phage, and the elimination of competing organisms, as well as those involved in facilitating the transfer of genetic material and protein to other bacteria, targeting host cells, and modulating host immune responses. This study provides a global view of native bacterial OMVs. This information will help us not only to elucidate the biogenesis and functions of OMV from nonpathogenic and pathogenic bacteria but also to develop vaccines and antibiotics effective against pathogenic strains.     

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.     

Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis.     

Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.     

Environmentally controlled bacterial vesicle‐mediated export

Over the past two decades, researchers studying both microbial and host cell communities have gained an appreciation for the ability of bacteria to produce, regulate, and functionally utilize outer membrane vesicles (OMVs) as a means to survive and interact with their cellular and acellular environments. Common ground has emerged, as it appears that vesicle production is an environmentally controlled and specific secretion process; however, it has been challenging to discover the principles that govern fundamentals of vesicle‐mediated transport. Namely, there does not appear to be a single mechanism modulating OMV export, nor universal “markers” for OMV cargo incorporation, nor particular host cell responses common to treatment with all OMVs. Given the diversity of species studied, their differences in envelope architecture and composition, the diversity of environmentally regulated bacterial processes, and the variety of interactions between bacteria and their abiotic and biotic environments, this is hardly surprising. Nevertheless, the ability of bacteria to control exported material in the context of a packaged insoluble particle, a vesicle, is emerging as a significant contribution to bacterial viability, biofilm communities, and bacterial‐host interactions. In this review, we focus on detailing important, recent findings regarding the content and functional differences in bacterially secreted vesicles that are influenced by growth conditions.     

LPS targets host guanylate‐binding proteins to the bacterial outer membrane for non‐canonical inflammasome activation

Pathogenic and commensal Gram‐negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase‐11 non‐canonical inflammasome. Here, we show that OMV‐induced inflammasome activation requires TLR4‐TRIF signaling, the production of type I interferons, and the action of guanylate‐binding proteins (GBPs), both in macrophages and in vivo. Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram‐negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell‐autonomous immunity and innate immunity.     

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1‐independent manner

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV‐associated CT is likewise trafficked to host cells in a GM1‐dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV‐associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1‐independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well‐studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1‐independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.