Extracellular Vesicles and Bacteriophages: New Directions in Environmental Biocolloid Research.

There is a long-standing appreciation among environmental engineers and scientists regarding the importance of biologically derived colloidal particles and their environmental fate. This interest has been recently renewed in considering bacteriophages and extracellular vesicles, which are each poised to offer engineers unique insights into fundamental aspects of environmental microbiology and novel approaches for engineering applications, including advances in wastewater treatment and sustainable agricultural practices. Challenges persist due to our limited understanding of interactions between these nanoscale particles with unique surface properties and their local environments. This review considers these biological particles through the lens of colloid science with attention given to their environmental impact and surface properties. We discuss methods developed for the study of inert (nonbiological) particle-particle interactions and the potential to use these to advance our understanding of the environmental fate and transport of extracellular vesicles and bacteriophages.

Comparative electrokinetic properties of extracellular vesicles produced by yeast and bacteria.

Extracellular vesicles (EVs) are nano-sized, biocolloidal proteoliposomes that have been shown to be produced by all cell types studied to date and are ubiquitous in the environment. Extensive literature on colloidal particles has demonstrated the implications of surface chemistry on transport behavior. Hence, one may anticipate that physicochemical properties of EVs, particularly surface charge-associated properties, may influence EV transport and specificity of interactions with surfaces. Here we compare the surface chemistry of EVs as expressed by zeta potential (calculated from electrophoretic mobility measurements). The zeta potentials of EVs produced by Pseudomonas fluorescens, Staphylococcus aureus, and Saccharomyces cerevisiae were largely unaffected by changes in ionic strength and electrolyte type, but were affected by changes in pH. The addition of humic acid altered the calculated zeta potential of the EVs, especially for those from S. cerevisiae. Differences in zeta potential were compared between EVs and their respective parent cell with no consistent trend emerging; however, significant differences were discovered between the different cell types and their EVs. These findings imply that, while EV surface charge (as estimated from zeta potential) is relatively insensitive to the evaluated environmental conditions, EVs from different organisms can differ regarding which conditions will cause colloidal instability.

Proteomic Profiling Reveals Distinct Bacterial Extracellular Vesicle Subpopulations with Possibly Unique Functionality.

Bacterial outer membrane vesicles (OMVs) are 20- to 200-nm secreted packages of lipids, small molecules, and proteins that contribute to diverse bacterial processes. In plant systems, OMVs from pathogenic and beneficial strains elicit plant immune responses that inhibit seedling growth and protect against future pathogen challenge. Previous studies of OMV-plant interactions suggest functionally important differences in the protein composition of Pseudomonas syringae and Pseudomonas fluorescens OMVs, and that their composition and activity differ as a result of medium culture conditions. Here, we show that plant apoplast-mimicking minimal medium conditions impact OMV protein content dramatically in P. syringae but not in P. fluorescens relative to complete medium conditions. Comparative, 2-way analysis of the four conditions reveals subsets of proteins that may contribute to OMV-mediated bacterial virulence and plant immune activation as well as those involved in bacterial stress tolerance or adaptation to a beneficial relationship with plants. Additional localization enrichment analysis of these subsets suggests the presence of outer-inner membrane vesicles (OIMVs). Collectively, these results reveal distinct differences in bacterial extracellular vesicle cargo and biogenesis routes from pathogenic and beneficial plant bacteria in different medium conditions and point to distinct populations of vesicles with diverse functional roles. IMPORTANCE Recent publications have shown that bacterial vesicles play important roles in interkingdom communication between bacteria and plants. Indeed, our recently published data reveal that bacterial vesicles from pathogenic and beneficial strains elicit immune responses in plants that protect against future pathogen challenge. However, the molecules underlying these striking phenomena remain unknown. Our recent work indicated that proteins packaged in vesicles are critically important for vesicle-mediated seedling growth inhibition, often considered an indirect measure of plant immune activation. In this study, we characterize the protein cargo of vesicles from Pseudomonas syringae pathovar tomato DC3000 and Pseudomonas fluorescens from two different medium conditions and show that distinct subpopulations of vesicles contribute to bacterial virulence and stress tolerance. Furthermore, we reveal differences in how beneficial and pathogenic bacterial species respond to harsh environmental conditions through vesicle packaging. Importantly, we find that protein cargo implicates outer-inner membrane vesicles in bacterial stress responses, while outer membrane vesicles are packaged for virulence.

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.

Differential Packaging Into Outer Membrane Vesicles Upon Oxidative Stress Reveals a General Mechanism for Cargo Selectivity.

Selective cargo packaging into bacterial extracellular vesicles has been reported and implicated in many biological processes, however, the mechanism behind the selectivity has remained largely unexplored. In this study, proteomic analysis of outer membrane (OM) and OM vesicle (OMV) fractions from enterotoxigenic E. coli revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. Selectivity was determined to be independent of transcriptional regulation of the proteins upon oxidative stress and was validated using randomly selected protein candidates from the different cargo classes. Based on these data, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA constructs. Our study reveals a basic mechanism for cargo selectivity into OMVs that may be useful for the engineering of OMVs for future biotechnological applications.

Microbial vesicle-mediated communication: convergence to understand interactions within and between domains of life.

All cells produce extracellular vesicles (EVs). These biological packages contain complex mixtures of molecular cargo and have a variety of functions, including interkingdom communication. Recent discoveries highlight the roles microbial EVs may play in the environment with respect to interactions with plants as well as nutrient cycling. These studies have also identified molecules present within EVs and associated with EV surfaces that contribute to these functions. In parallel, studies of engineered nanomaterials have developed methods to track and model small particle behavior in complex systems and measure the relative importance of various surface features on transport and function. While studies of EV behavior in complex environmental conditions have not yet employed transdisciplinary approaches, it is increasingly clear that expertise from disparate fields will be critical to understand the role of EVs in these systems. Here, we outline how the convergence of biology, soil geochemistry, and colloid science can both develop and address questions surrounding the basic principles governing EV-mediated interkingdom interactions.

Protective plant immune responses are elicited by bacterial outer membrane vesicles.

Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.

Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock.

Inflammation associated with gram-negative bacterial infections is often instigated by the bacterial cell wall component lipopolysaccharide (LPS). LPS-induced inflammation and resulting life-threatening sepsis are mediated by the two distinct LPS receptors TLR4 and caspase-11 (caspase-4/-5 in humans). Whereas the regulation of TLR4 activation by extracellular and phago-endosomal LPS has been studied in great detail, auxiliary host factors that specifically modulate recognition of cytosolic LPS by caspase-11 are largely unknown. This study identifies autophagy-related and dynamin-related membrane remodeling proteins belonging to the family of Immunity-related GTPases M clade (IRGM) as negative regulators of caspase-11 activation in macrophages. Phagocytes lacking expression of mouse isoform Irgm2 aberrantly activate caspase-11-dependent inflammatory responses when exposed to extracellular LPS, bacterial outer membrane vesicles, or gram-negative bacteria. Consequently, Irgm2-deficient mice display increased susceptibility to caspase-11-mediated septic shock in vivo. This Irgm2 phenotype is partly reversed by the simultaneous genetic deletion of the two additional Irgm paralogs Irgm1 and Irgm3, indicating that dysregulated Irgm isoform expression disrupts intracellular LPS processing pathways that limit LPS availability for caspase-11 activation.

Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles.

Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-ß response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

Outer Membrane Vesiculation Facilitates Surface Exchange and In Vivo Adaptation of Vibrio cholerae.

Gram-negative bacteria release outer membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the outer membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of outer membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use outer membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection.

Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity.

Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds, and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP-expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP-expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both nonself P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.IMPORTANCE Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight into how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins.

The Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as "free" LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1ß (IL-1ß) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections.

Breaking the bilayer: OMV formation during environmental transitions.

Gram-negative bacteria maintain the barrier properties of the outer membrane (OM) in a wide array of physiological conditions despite their inability to degrade lipopolysaccharide (LPS) and protein material present in the outer leaflet of the OM. Through characterization of the native dynamics of outer membrane LPS change we recently described a mechanism in which these diderm organisms overcome this design flaw. In response to different environmental stimuli Salmonellaenterica modulates the export of specific structural variants of lipid A via outer membrane vesicles (OMVs). We proposed that the polymorphic model for regulation of membrane lipid content could largely account for the structural differences between secreted and retained lipid A species. However, differences in OMV production levels and size observed between environmental conditions remain unexplained. Further exploration into the relationship between OMV production level and content specificity may shed light onto the enigmatic mechanisms of OMV formation.

Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions.

The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell's successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions. IMPORTANCE: All Gram-negative bacteria alter the structural composition of LPS present in their OM in response to various environmental stimuli. We developed a system to track the native dynamics of lipid A change in Salmonella enterica serovar Typhimurium following an environmental shift to PhoP/Q- and PmrA/B-inducing conditions. We show that growth conditions influence OMV production, size, and lipid A content. We further demonstrate that the lipid A content of OMVs does not fit a stochastic model of content selection, revealing the significant retention of lipid A species containing covalent modifications that mask their 1- and 4'-phosphate moieties under host-like conditions. Furthermore, palmitoylation of the lipid A to form hepta-acylated species substantially increases the likelihood of its incorporation into OMVs. These results highlight a role for the OMV response in OM remodeling and maintenance processes in Gram-negative bacteria.

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.

A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.

BACKGROUND: Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. MATERIALS AND METHODS: A standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets. RESULTS: PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs. CONCLUSION: The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions.

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.

Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.

Modulation of bacterial outer membrane vesicle production by envelope structure and content.

BACKGROUND: Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of outer membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp. RESULTS: In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in "periplasmic pressure" resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture. CONCLUSIONS: From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.