Jeffrey W. Schertzer

Bacterial outer membrane vesicles in trafficking, communication and the host-pathogen interaction.

Vesicular transport is a central process in eukaryotes that was believed to be absent in bacteria. However, as our understanding of the communal and interactive lifestyles of bacteria has increased by leaps and bounds, we are now well positioned to appreciate the many ways that membrane trafficking impacts this domain of life as well. Nearly all Gram-negative organisms release outer membrane vesicles into their environment. In this communication, we discuss the nature of these vesicles, the roles they play in bacterial physiology, ecology and virulence, and what is known about how they are formed. These remarkable structures can be used to confuse, communicate or kill depending on the situation and unlocking the mechanisms behind their formation, loading and delivery could lead to effective treatments against many important bacterial pathogens.

Membrane Distribution of the Pseudomonas Quinolone Signal Modulates Outer Membrane Vesicle Production in Pseudomonas aeruginosa

ABSTRACT The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis. IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa. In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors. IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa. In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors.

Membrane mechanical properties of synthetic asymmetric phospholipid vesicles

Synthetic lipid vesicles have served as important model systems to study cellular membrane biology. Research has shown that the mechanical properties of bilayer membranes significantly affects their biological behavior. The properties of a lipid bilayer are governed by lipid acyl chain length, headgroup type, and the presence of membrane proteins. However, few studies have explored how membrane architecture, in particular trans-bilayer lipid asymmetry, influences membrane mechanical properties. In this study, we investigated the effects of lipid bilayer architecture (i.e. asymmetry) on the mechanical properties of biological membranes. This was achieved using a customized micropipette aspiration system and a novel microfluidic technique previously developed by our team for building asymmetric phospholipid vesicles with tailored bilayer architecture. We found that the bending modulus and area expansion modulus of the synthetic asymmetric bilayers were up to 50% larger than the values acquired for symmetric bilayers. This was caused by the dissimilar lipid distribution in each leaflet of the bilayer for the asymmetric membrane. To the best of our knowledge, this is the first report on the impact of trans-bilayer asymmetry on the area expansion modulus of synthetic bilayer membranes. Since the mechanical properties of bilayer membranes play an important role in numerous cellular processes, these results have significant implications for membrane biology studies.

Understanding and Exploiting Bacterial Outer Membrane Vesicles

The bacterial outer surface defines the interface between an organism and its environment and thus plays an important role in facilitating interactions between the two. The structures comprising the cellular envelope have received considerable research interest, but an emerging area aims to understand how and why Gram negative bacteria actively shed fragments of their outer membrane into the surrounding milieu. This phenomenon has been observed in nearly all Gram-negative organisms studied and has even been described as a dedicated secretion system. In this communication, we describe the history, structure, composition, functions and mechanism of formation of outer membrane vesicles. In addition, we discuss the promise of exploiting OMVs for pharmaceutical purposes and the early successes that have been seen with OMV vaccines.

Detection of outer membrane vesicles in Synechocystis PCC 6803.

It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnological importance. We detected extracellular proteins and lipids in cell-free supernatants derived from Synechocystis culture, yet the cytoplasmic and thylakoid membrane markers NADH oxidase and chlorophyll were absent. This indicated that the extracellular proteins and lipids derived from the outer membrane, and not from cell lysis. Furthermore, we identified spherical structures within the expected size range of OMVs in Synechocystis culture using scanning electron microscopy. Taken together, these results suggest that the repertoire of Gram-negative bacteria that are known to produce OMVs may be expanded to include Synechocystis PCC6803. Because of the considerable genetic characterization of Synechocystis in particular, our discovery has the potential to support novel biotechnological applications as well.

Reciprocal cross-species induction of outer membrane vesicle biogenesis via secreted factors

Delivery of cargo to target cells is fundamental to bacterial competitiveness. One important but poorly understood system, ubiquitous among Gram-negative organisms, involves packaging cargo into outer membrane vesicles (OMVs). These biological nanoparticles are involved in processes ranging from toxin delivery to cell-cell communication. Despite this, we know comparatively little about how OMVs are formed. Building upon the discovery that the Pseudomonas Quinolone Signal (PQS) stimulates OMV biogenesis in Pseudomonas aeruginosa, we proposed a model where PQS interacts with the outer membrane to induce curvature and ultimately OMV formation. Though this model is well supported in P. aeruginosa, it remained unclear whether other organisms produce similar compounds. Here we describe the development of a tightly controlled experimental system to test the interaction of bacterially-produced factors with target cells. Using this system, we show that multiple species respond to PQS by increasing OMV formation, that PQS accumulates in the induced vesicles, and that other bacteria secrete OMV-promoting factors. Analysis of induced vesicles indicates that recipient-mediated mechanisms exist to control vesicle size and that relatedness to the producer organism can dictate susceptibility to OMV-inducing compounds. This work provides evidence that small molecule induced OMV biogenesis is a widely conserved process and that cross-talk between systems may influence OMV production in neighboring bacteria.

Synthetic Asymmetric Vesicles Built Using Microfluidic Technology at High-Throughput

We report on a novel microfluidic strategy for building monodisperse asymmetric vesicles with customized composition, size, and interfacial properties at high-throughput. The microfluidic device encompasses a triangular post region and two flow-focusing regions. The major steps involved in the vesicle building process include: (1) forming highly uniform water emulsion templates in the inner-leaflet lipid solution, (2) replacing the inner-leaflet lipid solution with the outer-leaflet lipid solution, (3) creating water-in-oil-in-water double emulsions, and (4) extracting the excess outer-leaflet lipid solution from the double emulsions. Bilayer membrane asymmetry and unilamellarity are confirmed using a fluorescence quenching assay and quantitative measurements of fluorescent intensities. This method addresses many of the deficiencies found in existing technologies, and yields asymmetries as high as 95%. The asymmetric vesicles built using this strategy hold the potential to serve as model systems to investigate fundamental problems in membrane biology.Copyright

Particulate and Emulsion Sorting Using Microfluidics

We report on a microfluidic device capable of sorting nanoscale particulates and water-in-oil emulsions at high-throughput. The device is passive, relying solely on hydrodynamic forces and the emulsion mass to achieve separation. We use the microfluidic device to deliver surfactants and lipids to the emulsion surface. This is achieved by immersing the emulsions in a fluid stream with a high concentration of the nano-particulates. The particulates assemble on the surface of the emulsions as they are transported along the stream. The emulsions are then transferred (i.e. separated) into a second fluid stream that is devoid of surrounding material. The performance of the device is evaluated for a range of flow rates, nano-particulate concentrations, and emulsion sizes. We report separation efficiencies that exceed current technologies over a wide range of flow rates. The microfluidic device can be used to produce delivery vehicles for pharmaceuticals and models for membrane biology studies.Copyright