Evolutionary loss of an antibiotic efflux pump increases quorum sensing mediated virulence .

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 () antibiotic efflux pump . Unexpectedly, efflux pump mutations increased 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.

Cytotoxic rhamnolipid micelles drive acute virulence in .

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, 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.

OprF functions as a latch to direct Outer Membrane Vesicle release in .

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.

Cytotoxic rhamnolipid micelles drive acute virulence in .

is an opportunistic human pathogen that has developed multi- or even pan-drug resistance towards most frontline and last resort antibiotics, leading to increasing infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, 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.

Effect of Local Stress on Accurate Modeling of Bacterial Outer Membranes Using All-Atom Molecular Dynamics.

Biological membranes are fundamental components of living organisms that play an undeniable role in their survival. Molecular dynamics (MD) serves as an essential computational tool for studying biomembranes on molecular and atomistic scales. The status quo of MD simulations of biomembranes studies a nanometer-sized membrane patch periodically extended under periodic boundary conditions (PBCs).

An integrated microfluidic platform to fabricate single-micrometer asymmetric giant unilamellar vesicles (GUVs) using dielectrophoretic separation of microemulsions.

We present a microfluidic technique that generates asymmetric giant unilamellar vesicles (GUVs) in the size range of 2-14 m. 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.

Quinolone Signal-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa.

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.

Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition.

The double-membrane cellular envelope of Gram-negative bacteria enables them to endure harsh environments and represents a barrier to many clinically available antibiotics. The outer membrane (OM) is exposed to the environment and is the first point of contact involved in bacterial processes such as signaling, pathogenesis, and motility. As in the cytoplasmic membrane, the OM in Gram-negative bacteria has a phospholipid-rich inner leaflet and an outer leaflet that is predominantly composed of lipopolysaccharide (LPS).

Analysis of Pseudomonas aeruginosa biofilm membrane vesicles supports multiple mechanisms of biogenesis.

Outer Membrane Vesicles (OMVs) are ubiquitous in bacterial environments and enable interactions within and between species. OMVs are observed in lab-grown and environmental biofilms, but our understanding of their function comes primarily from planktonic studies. Planktonic OMVs assist in toxin delivery, cell-cell communication, horizontal gene transfer, small RNA trafficking, and immune system evasion.

Molecular conformation affects the interaction of the quinolone signal with the bacterial outer membrane.

Gram-negative bacteria produce outer-membrane vesicles (OMVs) that package genetic elements, virulence factors, and cell-to-cell communication signaling compounds. Despite their importance in many disease-related processes, how these versatile structures are formed is incompletely understood. A self-produced secreted small molecule, the quinolone signal (PQS), has been shown to initiate OMV formation in by interacting with the outer membrane (OM) and inducing its curvature.

Genomic and Phenotypic Diversity among Ten Laboratory Isolates of PAO1.

is an opportunistic pathogen found ubiquitously in the environment and commonly associated with airway infection in patients with cystic fibrosis. strain PAO1 is one of the most commonly used laboratory-adapted research strains and is a standard laboratory-adapted strain in multiple laboratories and strain banks worldwide. Due to potential isolate-to-isolate variability, we investigated the genomic and phenotypic diversity among 10 PAO1 strains (henceforth called sublines) obtained from multiple research laboratories and commercial sources.

Molecular dynamics modeling of Pseudomonas aeruginosa outer membranes.

Pseudomonas aeruginosa is a common Gram-negative bacterium and opportunistic human pathogen. The distinctive structure of its outer membrane (OM) and outer membrane vesicles (OMVs) plays a fundamental role in bacterial virulence, colonization ability, and antibiotic resistance. To provide critical insights into OM and OMV functionality, we conducted an all-atom molecular dynamics study of asymmetric membranes that are biologically relevant to P.

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.

Membrane Distribution of the Quinolone Signal Modulates Outer Membrane Vesicle Production in .

The quinolone signal (PQS) is an important quorum-sensing molecule in 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.

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.

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.

Continuous microfluidic fabrication of synthetic asymmetric vesicles.

We report on a novel microfluidic strategy for the continuous fabrication of monodisperse asymmetric vesicles with customized membrane composition, size, and luminal content. The microfluidic device encompasses a triangular post region and two flow-focusing regions. The major steps involved in the vesicle fabrication process include: (1) forming highly uniform water emulsions in an oil/inner-leaflet-lipid solution, (2) replacing the inner-leaflet-lipid solution with an outer-leaflet-lipid solution inside the microchannel network, (3) forming water-in-oil-in-water double emulsions, and (4) extracting excess oil/outer-leaflet-lipid solution from the double emulsions.

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.

A bilayer-couple model of bacterial outer membrane vesicle biogenesis.

Unlabelled: Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated.

Microbial communication superhighways.

Exchange of information is critical for bacterial social behaviors. Now Dubey and Ben-Yehuda (2011) provide evidence for bacterial "nanotube" conduits that allow microbes to directly exchange cytoplasmic factors. Protein and DNA transfer between distantly related species raises the prospect of a new, widely distributed mechanism of bacterial communication.

Quantifying Pseudomonas aeruginosa quinolones and examining their interactions with lipids.

Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quantifying PQS. First, extraction of PQS from complex mixtures (e.

Oxygen levels rapidly modulate Pseudomonas aeruginosa social behaviours via substrate limitation of PqsH.

Many bacteria use extracellular signals to coordinate group behaviours, a process referred to as quorum sensing (QS). The bacterium Pseudomonas aeruginosa utilizes a complex QS system to control expression of over 300 genes, including many involved in host colonization and disease. The Pseudomonas quinolone signal (PQS) is a component of P.

More than a signal: non-signaling properties of quorum sensing molecules.

Quorum sensing in bacteria serves as an example of the adaptation of single-celled organisms to engage in cooperative group behaviors. This phenomenon is much more widespread than originally thought, with many different species 'speaking' through various secreted small molecules. Despite some variation in signaling molecules, the principles of quorum sensing are conserved across a wide range of organisms.

Use of CDP-glycerol as an alternate acceptor for the teichoic acid polymerase reveals that membrane association regulates polymer length.

The study of bacterial extracellular polysaccharide biosynthesis is hampered by the fact that these molecules are synthesized on membrane-resident carrier lipids. To get around this problem, a practical solution has been to synthesize soluble lipid analogs and study the biosynthetic enzymes using a soluble system. This has been done for the Bacillus subtilis teichoic acid polymerase, TagF, although several aspects of catalysis were inconsistent with the results obtained with reconstituted membrane systems or physiological observations.

Two conserved histidine residues are critical to the function of the TagF-like family of enzymes.

The TagF protein from Bacillus subtilis 168 is the poly(glycerol phosphate) polymerase responsible for the synthesis of wall teichoic acid and is the prototype member of a poorly understood family of similar teichoic acid synthetic enzymes. Here we describe in vitro and in vivo characterization of TagF, which localizes the active site to the carboxyl terminus of the protein and identifies residues that are critical for catalysis. We also establish the first mechanistic link among TagF and similar proteins by demonstrating that the identified residues are also critical in the function of TagB, a homologous enzyme implicated as the glycerophosphotransferase responsible for priming poly(glycerol phosphate) synthesis.