Cooperative Adsorption and Diffusion of Small Alcohols in Metal-Organic Framework ZIF-8 and Intrinsically Microporous Polymer PTMSP.

Fundamental understanding of molecular interactions and transport within microporous materials displaying cooperative Type V adsorption is challenged by the unique features of this isotherm type. In order to capture a broad understanding of this uncommon, yet industrially relevant, behavior in microporous materials, this investigation examines the adsorption equilibria and kinetics of methanol and ethanol in both a metal-organic framework (MOF) material, ZIF-8, and a high free volume polymer of intrinsic microporosity, poly[1-(trimethylsilyl)-1-propyne] (PTMSP). A novel formulation that can capture the cooperative effects of small alcohols in its description of adsorption equilibria and kinetics is proposed.

Potent activity of polymyxin B is associated with long-lived super-stoichiometric accumulation mediated by weak-affinity binding to lipid A.

Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model.

Alternative therapeutic strategies to treat antibiotic-resistant pathogens.

Resistance threatens to render antibiotics - which are essential for modern medicine - ineffective, thus posing a threat to human health. The discovery of novel classes of antibiotics able to overcome resistance has been stalled for decades, with the developmental pipeline relying almost entirely on variations of existing chemical scaffolds. Unfortunately, this approach has been unable to keep pace with resistance evolution, necessitating new therapeutic strategies.

Inhibitors targeting BamA in gram-negative bacteria.

Antibiotic resistance has led to an increase in the number of patient hospitalizations and deaths. The situation for gram-negative bacteria is especially dire as the last new class of antibiotics active against these bacteria was introduced to the clinic over 60 years ago, thus there is an immediate unmet need for new antibiotic classes able to overcome resistance. The outer membrane, a unique and essential structure in gram-negative bacteria, contains multiple potential antibacterial targets including BamA, an outer membrane protein that folds and inserts transmembrane β-barrel proteins.

Returning to Nature for the Next Generation of Antimicrobial Therapeutics.

Antibiotics found in and inspired by nature are life-saving cures for bacterial infections and have enabled modern medicine. However, the rise in resistance necessitates the discovery and development of novel antibiotics and alternative treatment strategies to prevent the return to a pre-antibiotic era. Once again, nature can serve as a source for new therapies in the form of natural product antibiotics and microbiota-based therapies.

Physical properties of the bacterial outer membrane.

It has long been appreciated that the Gram-negative outer membrane acts as a permeability barrier, but recent studies have uncovered a more expansive and versatile role for the outer membrane in cellular physiology and viability. Owing to recent developments in microfluidics and microscopy, the structural, rheological and mechanical properties of the outer membrane are becoming apparent across multiple scales. In this Review, we discuss experimental and computational studies that have revealed key molecular factors and interactions that give rise to the spatial organization, limited diffusivity and stress-bearing capacity of the outer membrane.

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.

The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity and is bactericidal against wild-type Acinetobacter baumannii and E.

Border Control: Regulating LPS Biogenesis.

The outer membrane (OM) is a defining feature of Gram-negative bacteria that serves as a permeability barrier and provides rigidity to the cell. Critical to OM function is establishing and maintaining an asymmetrical bilayer structure with phospholipids in the inner leaflet and the complex glycolipid lipopolysaccharide (LPS) in the outer leaflet. Cells ensure this asymmetry by regulating the biogenesis of lipid A, the conserved and essential anchor of LPS.

Structure of the essential inner membrane lipopolysaccharide-PbgA complex.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete.

Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of .

The Escherichia coli β-Barrel Assembly Machinery Is Sensitized to Perturbations under High Membrane Fluidity.

Integral β-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the β-barrel assembly machine (BAM). This essential complex, composed of a β-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of β-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function.

A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition.

Monoclonal antibody targeting the β-barrel assembly machine of is bactericidal.

The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the β-barrel assembly machine, BamA.

A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability.

Comprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing.

Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of ∼ 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation.

A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA.

Quorum sensing regulates the osmotic stress response in Vibrio harveyi.

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress.

Functional determinants of the quorum-sensing non-coding RNAs and their roles in target regulation.

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1-5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs.

Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation.

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins.

Individual and combined roles of the master regulators AphA and LuxR in control of the Vibrio harveyi quorum-sensing regulon.

Bacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors, AphA and LuxR, coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they coregulate 77 genes.

Bacterial quorum sensing: its role in virulence and possibilities for its control.

Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly. This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria.

AphA and LuxR/HapR reciprocally control quorum sensing in vibrios.

Bacteria cycle between periods when they perform individual behaviors and periods when they perform group behaviors. These transitions are controlled by a cell-cell communication process called quorum sensing, in which extracellular signal molecules, called autoinducers (AIs), are released, accumulate, and are synchronously detected by a group of bacteria. AI detection results in community-wide changes in gene expression, enabling bacteria to collectively execute behaviors such as bioluminescence, biofilm formation, and virulence factor production.