Jeffrey W. Schertzer

Bacterial outer membrane vesicles (OMVs) contribute to virulence, competition, immune avoidance, and communication. This has led to great interest in how they are formed. To date, investigation has focused almost exclusively on what controls the initiation of OMV biogenesis. Regardless of the mechanism of initiation, all species face a similar challenge before an OMV can be released: how does the outer membrane (OM) detach from the underlying peptidoglycan (PG) in regions that will ultimately bulge and then vesiculate? The OmpA family of OM proteins (OprF in Pseudomonas aeruginosa ) is widely conserved and unusually abundant in OMVs across species, considering their major role in PG attachment. OmpA homologs also have the interesting ability to adopt both PG-bound (two-domain) and PG-released (one-domain) conformations. Using targeted deletion of the PG-binding domain, we show here that loss of cell wall association, not general membrane destabilization, is responsible for hypervesiculation in OprF-modified strains. We therefore propose that OprF functions as a “latch,” capable of releasing PG in regions destined to become OMVs. To test this hypothesis, we developed a protocol to assess OprF conformation in live cells and purified OMVs. While >90% of OprF proteins exist in the two-domain conformation in the OM of cells, we show that the majority of OprF in OMVs is present in the one-domain conformation. With this work, we take some of the first steps in characterizing late-stage OMV biogenesis and identify a family of proteins whose critical role can be explained by their unique ability to fold into two distinct conformations. IMPORTANCE: Vesicular transport is now recognized to operate in all domains of life. Nevertheless, the study of outer membrane vesicle (OMV) biogenesis has been challenging because genetic screens failed to identify proteins analogous to those involved in eukaryotic vesicular transport. With this work, we identify the first protein whose direct action can both define the location and govern the mechanism of OMV release. Our latch model is consistent with previous observations linking OmpA family proteins to OMV biogenesis but further describes a physical mechanism that has broad implications for vesicle production and function across species. The work presented here unifies disparate OMV biogenesis mechanisms at the point of vesicle release and underscores the stark differences in how transport vesicles are formed in prokaryotes vs eukaryotes.

The ability to use vesicles to deliver cargo safely across distance is a valuable strategy that appears to be universal across all life. The ability of prokaryotes to accomplish this is a relatively new idea, but one that is expanding rapidly. In this review, we will describe what bacterial extracellular vesicles (bEVs) are and where they come from. We will also summarize what has been learned about their biogenesis and function through foundational planktonic studies. Using this as a backdrop, we will then contextualize new and exciting discoveries made as the focus of the field has shifted to studying bEVs in more complex multi-species and biofilm communities. We will explore how, when and where bEVs are produced in biofilms and how the circumstances of their formation can affect communication, matrix remodeling, community metabolism, organization, and defense. The boundless versatility of bEVs makes them essential contributors to bacterial community life and also offers great potential for exploitation as applied biologics.

Antibiotic resistance is a threat to human health, yet recent work highlights how loss of resistance may drive pathogenesis in some bacteria. In two recent studies, we found that β-lactam antibiotics and nutrient stresses faced during infection selected for genetic inactivation of the Pseudomonas aeruginosa antibiotic efflux pump mexEFoprN. Unexpectedly, efflux pump mutations increased P. aeruginosa virulence during infection; however, neither the prevalence of mexEFoprN inactivating mutations in real human infections, nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for virulence enhancing mutations. Using genome sequencing of clinical isolates, we show that mexEFoprN efflux pump inactivating mutations are enriched in P. aeruginosa isolates from cystic fibrosis infections relative to isolates from acute respiratory infections. Combining RNA-seq, metabolomics, genetic approaches, and infection models we show that efflux pump mutants have elevated quorum sensing driven expression of elastase and rhamnolipids which increase P. aeruginosa virulence during acute and chronic infections. Restoration of the efflux pump in a representative respiratory isolate and the notorious cystic fibrosis Liverpool epidemic strain reduced their virulence. These findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity.

Gram-negative bacteria use outer membrane vesicles (OMVs) for toxin trafficking, immune interference, horizontal gene transfer, antibiotic protection, and cell–cell communication. Despite their direct contribution to many pathogenesis-related behaviors, our understanding of how OMVs are produced remains surprisingly incomplete. The Bilayer Couple model describes the induction of OMV formation resulting from the preferential accumulation of small molecules in the outer leaflet of the membrane, resulting in leaflet expansion and membrane bending. Previous work has highlighted the importance of the structure of the Pseudomonas Quinolone Signal (PQS) in driving OMV formation, but the nature of interactions with membrane lipids remains unclear. Our recent in silico analysis suggested that a new interaction, between the PQS ring nitrogen and Lipid A, is critical for PQS function. Here, we used chemical analogs to interrogate the importance of specific PQS functional groups in its ability to stimulate OMV biogenesis. We demonstrated that OMV induction requires the presence of all PQS functional groups together. Further modeling uncovered that PQS prefers interaction with the outer leaflet of the membrane, consistent with its unique ability to drive OMV biogenesis. This was explained by much greater hydrogen bond formation between PQS and Lipid A. Interestingly, the preference of PQS for the outer leaflet coincided with that leaflet becoming crowded. Thus, the initial insertion of PQS into the outer leaflet would be expected to encourage local accumulation of more PQS to drive the induction of membrane curvature and subsequent OMV formation.

Antibiotic resistance is one of the most pressing threats to human health, yet recent work highlights how loss of resistance may also drive pathogenesis in some bacteria. In two recent studies, we found that β-lactam antibiotic and nutrient stresses faced during infection selected for the genetic inactivation of the Pseudomonas aeruginosa (Pa) antibiotic efflux pump mexEFoprN. Unexpectedly, efflux pump mutations increased Pa virulence during infection; however, neither the prevalence of efflux pump inactivating mutations in real human infections, nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for efflux pump mutations that drive increased virulence in Pa clinical isolates. Using genome sequencing of hundreds of Pa clinical isolates, we show that mexEFoprN efflux pump inactivating mutations are enriched in Pa cystic fibrosis isolates relative to Pa intensive care unit clinical isolates. Combining RNA-seq, metabolomics, genetic approaches, and infection models we show that efflux pump mutants have elevated expression of two key Pa virulence factors, elastase and rhamnolipids, which increased Pa virulence and lung damage during both acute and chronic infections. Increased virulence factor production was driven by higher Pseudomonas quinolone signal levels in the efflux pump mutants. Finally, genetic restoration of the efflux pump in a representative ICU clinical isolate and the notorious CF Pa Liverpool epidemic strain reduced their virulence. Together, our findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed multi- or even pan-drug resistance toward most frontline and last resort antibiotics, leading to increasing frequency of infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, P. aeruginosa produces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity toward murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even noncellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated with P. aeruginosa virulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion during P. aeruginosa infections.

Bacterial Outer Membrane Vesicles (OMVs) contribute to virulence, competition, immune avoidance and communication. This has led to great interest in how they are formed. To date, investigation has focused almost exclusively on what controls the initiation of OMV biogenesis. Regardless of the mechanism of initiation, all species face a similar challenge before an OMV can be released: How does the OM detach from the underlying peptidoglycan (PG) in regions that will ultimately bulge and then vesiculate? The OmpA family of OM proteins (OprF in P. aeruginosa ) is widely conserved and unusually abundant in OMVs across species considering their major role in PG attachment. OmpA homologs also have the interesting ability to adopt both PG-bound (two-domain) and PG-released (one-domain) conformations. Using targeted deletion of the PG-binding domain we showed that loss of cell wall association, and not general membrane destabilization, is responsible for hypervesiculation in OprF-modified strains. We therefore propose that OprF functions as a ‘latch’, capable of releasing PG in regions destined to become OMVs. To test this hypothesis, we developed a protocol to assess OprF conformation in live cells and purified OMVs. While >90% of OprF proteins exist in the two-domain conformation in the OM of cells, we show that the majority of OprF in OMVs is present in the one-domain conformation. With this work, we take some of the first steps in characterizing late-stage OMV biogenesis and identify a family of proteins whose critical role can be explained by their unique ability to fold into two distinct conformations. Significance: Vesicular transport is now recognized to operate in all domains of life. However, the study of OMV biogenesis has been challenging because genetic screens failed to identify proteins analogous to those involved in eukaryotic vesicular transport. With this work we identify the first protein whose direct action can both define the location and govern the mechanism of OMV release. Our latch model is consistent with previous observations linking OmpA family proteins to OMV biogenesis, but further describes a physical mechanism that has broad implications for vesicle production and function across species. The work presented here advances our understanding of a fundamental virulence-associated process in bacteria, while underscoring stark differences in how transport vesicles are formed in prokaryotes versus eukaryotes.

We present a microfluidic technique that generates asymmetric giant unilamellar vesicles (GUVs) in the size range of 2–14 μm. In our method, we (i) create water-in-oil emulsions as the precursors to build synthetic vesicles, (ii) deflect the emulsions across two oil streams containing different phospholipids at high throughput to establish an asymmetric architecture in the lipid bilayer membranes, and (iii) direct the water-in-oil emulsions across the oil–water interface of an oscillating oil jet in a co-flowing confined geometry to encapsulate the inner aqueous phase inside a lipid bilayer and complete the fabrication of GUVs. In the first step, we utilize a flow-focusing geometry with precisely controlled pneumatic pressures to form monodisperse water-in-oil emulsions. We observed different regimes in forming water-in-oil multiphase flows by changing the applied pressures and discovered a hysteretic behavior in jet breakup and droplet generation. In the second step of GUV fabrication, an oil stream containing phospholipids carries the emulsions into a separation region where we steer the emulsions across two parallel oil streams using active dielectrophoretic and pinched-flow fractionation separations. We explore the effect of applied DC voltage magnitude and carrier oil stream flow rate on the separation efficiency. We develop an image processing code that measures the degree of mixing between the two oil streams as the water-in-oil emulsions travel across them under dielectrophoretic steering to find the ideal operational conditions. Finally, we utilize an oscillating co-flowing jet to complete the formation of asymmetric giant unilamellar vesicles and transfer them to an aqueous phase. We investigate the effect of flow rates on properties of the co-flowing jet oscillating in the whipping mode (i.e., wavelength and amplitude) and define the phase diagram for the oil-in-water jet. Assays used to probe the lipid bilayer membrane of fabricated GUVs showed that membranes were unilamellar, minimal residual oil remained trapped between the two lipid leaflets, and 83% asymmetry was achieved across the lipid bilayers of GUVs.

Bacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of antibiofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital-acquired infections and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-cell communication, virulence factor delivery, and transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Several groups have shown that Pseudomonas quinolone signal (PQS) induces OMV formation in P. aeruginosa. Our group described a biophysical mechanism for this and recently showed it is operative in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype was rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components. IMPORTANCE Treatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. Pseudomonas quinolone signal (PQS) has been implicated in early biofilm development, but we hypothesized that its function as an outer membrane vesicle (OMV) inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and provide evidence that effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. These findings lay the groundwork for understanding OMV contributions to biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.