Gram-negative Outer Membrane Research Articles

YhdP, TamB, and YdbH Are Redundant but Essential for Growth and Lipid Homeostasis of the Gram-Negative Outer Membrane.

ABSTRACTThe bacterial cell envelope is the first line of defense and point of contact with the environment and other organisms. Envelope biogenesis is therefore crucial for the survival and physiology of bacteria and is often targeted by antimicrobials. Gram-negative bacteria have a multilayered envelope delimited by an inner and outer membrane (IM and OM, respectively). The OM is a barrier against many antimicrobials because of its asymmetric lipid structure, with phospholipids composing the inner leaflet and lipopolysaccharides (LPS) the outer leaflet. Since lipid synthesis occurs at the IM, phospholipids and LPS are transported across the cell envelope and asymmetrically assembled at the OM during growth. How phospholipids are transported to the OM remains unknown. Recently, the Escherichia coli protein YhdP has been proposed to participate in this process through an unknown mechanism. YhdP belongs to the AsmA-like clan and contains domains homologous to those found in lipid transporters. Here, we used genetics to investigate the six members of the AsmA-like clan of proteins in E. coli. Our data show that YhdP and its paralogs TamB and YdbH are redundant, but not equivalent, in performing an essential function in the cell envelope. Among the AsmA-like paralogs, only the combined loss of YhdP, TamB, and YdbH is lethal, and any of these three proteins is sufficient for growth. We also show that these proteins are required for OM lipid homeostasis and propose that they are the long-sought-after phospholipid transporters that are required for OM biogenesis.

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. These physical properties suggest broad connections between cellular structure and physiology, and we explore future prospects for further elucidation of the implications of outer membrane construction for cellular fitness and survival.

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer.

Gram-negative bacteria release Outer Membrane Vesicles (OMVs) into the extracellular environment. Recent studies recognized these vesicles as vectors to horizontal gene transfer; however, the parameters that mediate OMVs transfer within bacterial communities remain unclear. The present study highlights for the first time the transfer of plasmids containing resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). This mechanism confers DNA protection, it is plasmid copy number dependent with a ratio of 3.6 times among high copy number plasmid (pGR) versus low copy number plasmid (PRM), and the transformation efficiency was 3.6 times greater. Therefore, the DNA amount in the vesicular lumen and the efficacy of horizontal gene transfer was strictly dependent on the identity of the plasmid. Moreover, the role of K. pneumoniae-OMVs in interspecies transfer was described. The transfer ability was not related to the phylogenetic characteristics between the donor and the recipient species. K. pneumoniae-OMVs transferred plasmid to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Burkholderia cepacia. These findings address the pivotal role of K. pneumoniae-OMVs as vectors for antimicrobial resistance genes spread, contributing to the development of antibiotic resistance in the microbial communities.

A Convenient Chemoenzymatic Preparation of Chimeric Macrocyclic Peptide Antibiotics with Potent Activity against Gram-Negative Pathogens.

The continuing rise of antibiotic resistance, particularly among Gram-negative pathogens, threatens to undermine many aspects of modern medical practice. To address this threat, novel antibiotics that utilize unexploited bacterial targets are urgently needed. Over the past decade, a number of studies have highlighted the antibacterial potential of macrocyclic peptides that target Gram-negative outer membrane proteins (OMPs). Recently, it was reported that the antibacterial activities of OMP-targeting macrocyclic peptidomimetics of the antimicrobial peptide protegrin-1 are dramatically enhanced upon linking to polymyxin E nonapeptide (PMEN). In this study, we describe a convergent, chemoenzymatic route for the convenient preparation of such conjugates. Specifically, we investigated the use of both amide bond formation and azide-alkyne ligation for connecting an OMP-targeting macrocyclic peptide to a PMEN building block (obtained by enzymatic degradation of polymyxin E). The conjugates obtained via both approaches display potent antibacterial activity against a range of Gram-negative pathogens including multi-drug-resistant isolates.

Rifabutin for infusion (BV100) for the treatment of severe carbapenem-resistant Acinetobacter baumannii infections

Rifamycin antibiotics were discovered during the 1950s, and their main representative, rifampicin, remains a cornerstone treatment for TB. The clinical use of rifamycin is restricted to mycobacteria and Gram-positive infections because of its poor ability to penetrate the Gram-negative outer membrane. Rifabutin, a rifamycin antibiotic approved for the prevention of Mycobacterium avium complex disease, makes an exception to this rule by hijacking the iron uptake system of Acinetobacter baumannii, resulting in potent activity against this important Gram-negative pathogen. Here, we describe recent findings on the specific activity of rifabutin and provide evidence of the need for the development of an intravenous formulation of rifabutin (BV100) for the treatment of difficult-to-treat carbapenem-resistant A.baumannii infections.

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

ABSTRACTLipoprotein 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 in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport.IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

Cardiolipin aids in lipopolysaccharide transport to the gram-negative outer membrane

In Escherichia coli, cardiolipin (CL) is the least abundant of the three major glycerophospholipids in the gram-negative cell envelope. However, E. coli harbors three distinct enzymes that synthesize CL: ClsA, ClsB, and ClsC. This redundancy suggests that CL is essential for bacterial fitness, yet CL-deficient bacteria are viable. Although multiple CL-protein interactions have been identified, the role of CL still remains unclear. To identify genes that impact fitness in the absence of CL, we analyzed high-density transposon (Tn) mutant libraries in combinatorial CL synthase mutant backgrounds. We found LpxM, which is the last enzyme in lipid A biosynthesis, the membrane anchor of lipopolysaccharide (LPS), to be critical for viability in the absence of clsA Here, we demonstrate that CL produced by ClsA enhances LPS transport. Suppressors of clsA and lpxM essentiality were identified in msbA, a gene that encodes the indispensable LPS ABC transporter. Depletion of ClsA in ∆lpxM mutants increased accumulation of LPS in the inner membrane, demonstrating that the synthetic lethal phenotype arises from improper LPS transport. Additionally, overexpression of ClsA alleviated ΔlpxM defects associated with impaired outer membrane asymmetry. Mutations that lower LPS levels, such as a YejM truncation or alteration in the fatty acid pool, were sufficient in overcoming the synthetically lethal ΔclsA ΔlpxM phenotype. Our results support a model in which CL aids in the transportation of LPS, a unique glycolipid, and adds to the growing repertoire of CL-protein interactions important for bacterial transport systems.

A Screen of Natural Product Extracts Identifies Moenomycin as a Potent Antigonococcal Agent.

Increasing multidrug resistance in Neisseria gonorrheae is a growing public health crisis. Resistance to the last line therapies, cephalosporins and azithromycin, are of particular concern, fueling the need to discover new treatments. Here, we identified the phosphoglycolipid moenomycin from a screen of microbial natural products against drug-resistant N. gonorrheae as a potent antigonococcal agent. Moenomycin demonstrates excellent activity (MIC = 0.004-0.03 μg/mL) against a variety of multidrug-resistant N. gonorrheae. Importantly, moenomycin, thought to be a Gram-positive specific antibiotic, penetrates the Gram-negative gonococcal outer membrane. Moenomycin causes intracellular accumulation of peptidoglycan precursors, cell blebbing, and rupture of the cell envelope, all consistent with cell wall biosynthesis inhibition. Serial bacterial exposure to moenomycin for 14 days revealed slow development of resistance (MICDay14 = 0.03-0.06 μg/mL), unlike the clinically used drug azithromycin. Our results offer the potential utility of moenomycin as a lead for antigonococcal therapeutic candidates and warrant further investigation.

Thrombin-Derived Peptides Potentiate the Activity of Gram-Positive-Specific Antibiotics against Gram-Negative Bacteria.

The continued rise of antibiotic resistance threatens to undermine the utility of the world’s current antibiotic arsenal. This problem is particularly troubling when it comes to Gram-negative pathogens for which there are inherently fewer antibiotics available. To address this challenge, recent attention has been focused on finding compounds capable of disrupting the Gram-negative outer membrane as a means of potentiating otherwise Gram-positive-specific antibiotics. In this regard, agents capable of binding to the lipopolysaccharide (LPS) present in the Gram-negative outer membrane are of particular interest as synergists. Recently, thrombin-derived C-terminal peptides (TCPs) were reported to exhibit unique LPS-binding properties. We here describe investigations establishing the capacity of TCPs to act as synergists with the antibiotics erythromycin, rifampicin, novobiocin, and vancomycin against multiple Gram-negative strains including polymyxin-resistant clinical isolates. We further assessed the structural features most important for the observed synergy and characterized the outer membrane permeabilizing activity of the most potent synergists. Our investigations highlight the potential for such peptides in expanding the therapeutic range of antibiotics typically only used to treat Gram-positive infections.

Discovery of Macrolide Antibiotics Effective against Multi-Drug Resistant Gram-Negative Pathogens.

Macrolides are among the most widely prescribed antibiotics, particularly for bacterial lung infections, due to their favorable safety, oral bioavailability, and spectrum of activity against Gram-positive pathogens such as Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Their utility against Gram-negative bacteria is extremely limited and does not include the Enterobacteriaceae or other ESKAPE pathogens. With the increasing development of resistance to current therapies and the lack of safe, oral options to treat Gram-negative infections, extended-spectrum macrolides have the potential to provide valuable treatment options. While the bacterial ribosome, the target of macrolides, is highly conserved across Gram-positive and Gram-negative bacteria, traditional macrolides do not possess the proper physicochemical properties to cross the polar Gram-negative outer membrane and are highly susceptible to efflux. As with most natural product-derived compounds, macrolides are generally prepared through semisynthesis, which is limited in scope and lacks the ability to make the drastic physicochemical property changes necessary to overcome these hurdles.By using a fully synthetic platform technology to greatly expand structural diversity, novel macrolides were prepared with a focus on lowering the MW and increasing the polarity to achieve a physicochemical property profile more similar to that of traditional Gram-negative drug classes. In addition to the removal of lipophilic groups, a critical structural feature for obtaining Gram-negative activity in the macrolide class proved to be the introduction of small secondary or tertiary amines to yield polycationic species potentially capable of self-promoted uptake. Within the azithromycin-like 15-membered azalides, potent activity was seen when small alkyl amines were introduced at the 6'-position of desosamine. The biggest gains, however, were made by replacing the entire C10-C13 fragment of the macrolactone ring with commercially available or readily synthesized 1,2-aminoalcohols, leading to 13-membered azalides. The introduction of a tethered basic amine at the C10-position and systematic optimization of substitution and tether length and flexibility ultimately provided new macrolides that for the first time exhibit clinically relevant antibacterial activity against multi-drug resistant Gram-negative bacteria. A retrospective computational analysis of >1800 fully synthetic macrolides prepared during this effort identified key drivers and optimum ranges for improving permeability and avoiding efflux. In contrast to standard Gram-negative drugs which generally have MWs below 600 and clogD7.4 values below 0, we found that the ideal ranges for Gram-negative macrolides were MW between 600 and 720 and cLogD7.4 between -1 and 3. A total charge of between 2.5 and 3 was also required to provide optimal permeability and efflux avoidance. Thus, Gram-negative macrolides occupy a unique physicochemical property space that lies between traditional Gram-negative drug classes and Gram-positive macrolides.

Bacterial Mimetic Systems for Studying Bacterial Inactivation and Infection

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins and glycans, which forms a rigid protective layer against the environment. The major component of the outer membrane is the large and amphiphilic molecule lipopolysaccharide (LPS). Understanding the cell wall is important for various fields such as electric field treatments of bacteria and exploration of new antibiotics and alternative treatments (e.g. bacteriophage therapies). Due to the high complexity of the Gram-negative cell wall, there is a great demand for reduced and defined model membrane systems. Giant unilamellar vesicles (GUVs) can provide such a model membrane system. GUVs are versatile synthetic membrane systems that allow the investigation of membrane properties, while controlling parameters such as membrane composition, surrounding media and temperature. Furthermore, effects of external factors like ions, hydrodynamic flows and electromagnetic fields can be studied. In contrast to eukaryotic membranes, models of the Gram-negative outer membrane are lacking. Here, we aim at generating an in vitro model of the Gram-negative bacterial outer membrane. To create such a model membrane system, LPS are integrated in the GUV outer leaflet (LPS-GUVs) using an adapted method based on inverted emulsions. A microfluidic approach is employed to optimize production. The obtained LPS-GUVs will be characterized according to their LPS content, membrane asymmetry and membrane material properties such as bending rigidity and edge tension. Their stability against externally applied electric fields will be compared to that of conventional phospholipid vesicles. Furthermore, the interaction with LPS specific bacteriophages will be studied.

Towards Understanding Antibiotic Permeation Across The Gram-Negative Bacteria Outer Membrane

Towards Understanding Antibiotic Permeation Across The Gram-Negative Bacteria Outer Membrane

Gut Immunity and Microbiota Dysbiosis Are Associated with Altered Bile Acid Metabolism in LPS-Challenged Piglets.

Bacterial infections are among the major factors that cause stress and intestinal diseases in piglets. Lipopolysaccharide (LPS), a major component of the Gram-negative bacteria outer membrane, is commonly employed for inducing an immune response in normal organisms for convenience. The association between LPS stimulation and gut immunity has been reported. However, the effects of gut immunity on microbial homeostasis and metabolism of host, especially bile acid and lipid metabolism in piglets, remain unclear. Hence, in the current study, we elucidated the effect of gut immunity on microbial balance and host metabolism. Twenty-one-day-old healthy piglets (male) were randomly assigned into the CON and LPS groups. After 4 hours of treatment, related tissues and cecal contents were obtained for further analysis. The obtained results showed that stimulated LPS considerably damaged the morphology of intestinal villi and enhanced the relative expression of proinflammatory cytokines. Besides, LPS partially changed the microbial structure as indicated by β-diversity and increased operational taxonomic units (OTUs) related to Oxalobacter and Ileibacterium. Furthermore, bile acid, a large class of gut microbiota metabolites, was also assessed by many proteins related to the enterohepatic circulation of bile acids. It was also revealed that LPS markedly inhibited the mRNA and protein expression of TGR5 and FXR (bile acid receptors) in the ileum, which expressed negative feedback on bile acid de novo synthesis. Additionally, results indicated upregulated mRNA of genes associated with the production of bile acid in the liver tissues. Moreover, LPS reduced the expression of bile acid transporters in the ileum and liver tissues and further disturbed the normal enterohepatic circulation. Taken together, gut immunity and microbial dysbiosis are associated with altered bile acid metabolism in LPS-challenged piglets, which provided theoretical basis for revealing the potential mechanism of intestinal inflammation in swine and seeking nutrients to resist intestinal damage.

Biomimetic enterobactin analogue mediates iron-uptake and cargo transport into E. coli and P. aeruginosa.

The design, synthesis and biological evaluation of the artificial enterobactin analogue EntKL and several fluorophore-conjugates thereof are described. EntKL provides an attachment point for cargos such as fluorophores or antimicrobial payloads. Corresponding conjugates are recognized by outer membrane siderophore receptors of Gram-negative pathogens and retain the natural hydrolyzability of the tris-lactone backbone. Initial density-functional theory (DFT) calculations of the free energies of solvation (ΔG(sol)) and relaxed Fe–O force constants of the corresponding [Fe-EntKL]3− complexes indicated a similar iron binding constant compared to natural enterobactin (Ent). The synthesis of EntKL was achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an overall yield of 3%. A series of growth recovery assays under iron-limiting conditions with Escherichia coli and Pseudomonas aeruginosa mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of EntKL similar to natural Ent. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of E. coli indicating an uptake into the cytosol. P. aeruginosa displayed a stronger uptake promiscuity as six different cargo-conjugates were found to restore growth under iron-limiting conditions. Imaging studies utilizing BODIPYFL-conjugates, demonstrated the ability of EntKL to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of E. coli and P. aeruginosa.

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

Eucalyptus camaldulensis efficiency for application against microbial infections

On account of the continuing increase in antimicrobial resistance among infectious agents which are mostly life-threatening, seeking novel and alternative therapeutic approaches seems essential. Eucalyptus camaldulensis, generally known as eucalyptus, contains various healing traits such as antimicrobial characteristics. These herbs generally grow alongside the rivers with both continuous and seasonal water. The antimicrobial applications of this herb, particularly against pandrug and multidrug resistant microorganisms mostly attributed to phenolic, polar terpene compounds and spathulenol. Major bioactive antimicrobial fractions include flavonoids, alkaloids, pigments, terpenes, phenolics, starches, steroids and essential oils. E. camaldulensis has antimicrobial features against various bacterial (both Gram-positive and Gram-negative species), fungal, parasitic and viral infections. Regarding molecular mechanisms, the discharge of Gram-negative outer membrane lipopolysaccharide and increase the permeability of the cytoplasmic membrane have been proposed. Leaf extracts might be a good source of antibacterial and antifungal materials that might be useful in pharmaceutical industry. Interactions with various conventional antimicrobial agents providing synergistic effects suppose a variety of antimicrobial mechanisms by fractions remaining to be entirely elucidated.

Effect of extracted LPS of Salmonella typhimurium on chicken immunity.

In this study, the prevalence of Salmonella typhimurium in Egyptian chicken farms was studied. Examination of 79 chicken farms distributed in Egypt governorates from 2017 to 2019. Salmonella was detected in broiler flocks 20 % positivity (18 out of 90). However, in layer flocks showed 70 % positivity (7 out of 10). Then Salmonella typhimurium was confirmed using Salmonella typhimurium STM4 495 gene using conventional PCR. Lipopolysaccharides (LPSs) are the major constituent of the bacterial Gram-negative outer membrane including Salmonella. (LPSs) is an immunodominant molecule that is important for the virulence and pathogenesis of Salmonella typhimurium which called endotoxin. Extraction and purification of LPS from isolated S. typhimurium using isopropanol and sodium hydroxide mixture this overcome the presence of nucleic acids of extracted Salmonella in the final product. The purity of LPS isolated from Salmonella typhimurium was assessed by HPLC showing highly purified extracted LPS. Effect of extracted LPS of Salmonella typhimurium on poultry immunity were studied via a biological experiment using Chicks of the broiler breeds to assess the response to LPS stimulation via in vivo experiment using Different concentrations of extracted LPS (50,100,150 and 200 μg/kg) were injected subcutaneously in 21-day old SPF chicks. After 3 hours of injection samples taken to assest of the effect of LPS on RNA expression of the immune-related genes; interferon-γ (IFN-γ) and interleukin 1β (IL-1β). There was significant upregulation of IFN-γ and low downregulation of IL-1β of the immune-related genes.

Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations

Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-β-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM.

Studying the surfaces of bacteria using neutron scattering: finding new openings for antibiotics.

The use of neutrons as a scattering probe to investigate biological membranes has steadily grown in the past three decades, shedding light on the structure and behaviour of this ubiquitous and fundamental biological barrier. Meanwhile, the rise of antibiotic resistance has catalysed a renewed interest in understanding the mechanisms underlying the dynamics of antibiotics interaction with the bacterial cell envelope. It is widely recognised that the key reason behind the remarkable success of Gram-negative pathogens in developing antibiotic resistance lies in the effectiveness of their outer membrane (OM) in defending the cell from antibacterial compounds. Critical to its function, the highly asymmetric lipid distribution between the inner and outer bilayer leaflets of the OM, adds an extra level of complexity to the study of this crucial defence barrier. Here we review the opportunities offered by neutron scattering techniques, in particular reflectometry, to provide structural information on the interactions of antimicrobials with in vitro models of the OM. The differential sensitivity of neutrons towards hydrogen and deuterium makes them a unique probe to study the structure and behaviour of asymmetric membranes. Molecular-level understanding of the interactions between antimicrobials and the Gram-negative OM provides valuable insights that can aid drug development and broaden our knowledge of this critically important biological barrier.

Polymyxins and Bacterial Membranes: A Review of Antibacterial Activity and Mechanisms of Resistance.

Following their initial discovery in the 1940s, polymyxin antibiotics fell into disfavor due to their potential clinical toxicity, especially nephrotoxicity. However, the dry antibiotic development pipeline, together with the rising global prevalence of infections caused by multidrug-resistant (MDR) Gram-negative bacteria have both rejuvenated clinical interest in these polypeptide antibiotics. Parallel to the revival of their use, investigations into the mechanisms of action and resistance to polymyxins have intensified. With an initial known effect on biological membranes, research has uncovered the detailed molecular and chemical interactions that polymyxins have with Gram-negative outer membranes and lipopolysaccharide structure. In addition, genetic and epidemiological studies have revealed the basis of resistance to these agents. Nowadays, resistance to polymyxins in MDR Gram-negative pathogens is well elucidated, with chromosomal as well as plasmid-encoded, transferrable pathways. The aims of the current review are to highlight the important chemical, microbiological, and pharmacological properties of polymyxins, to discuss their mechanistic effects on bacterial membranes, and to revise the current knowledge about Gram-negative acquired resistance to these agents. Finally, recent research, directed towards new perspectives for improving these old agents utilized in the 21st century, to combat drug-resistant pathogens, is summarized.