Roflamycoin--a new channel-forming antibiotic

Ion permeability of lipid bilayers was studied in the presence of a new antifungal pentaene antibiotic, roflamycoin, the structure of which differs considerably from that of the well-known polyene channel-former amphotericin B. Both of them, however, show the property of increasing the membrane permeability only in the case of sterol-containing membrane when added on both its sides. The conductance is strongly dependent on the concentration of the antibiotic in the solutions and of sterol in the membrane. Unlike the amphotericin B channels, roflamycoin channels are potential-dependent and have short lifetime (approx. 1 s) and high conductance (approx. 100 ps in 1 M KCl), which increases linearly with the salt concentration and is not blocked by the familiar blockers of amphotericin B channels. The two antibiotics seem to have a common mechanism of channel formation, viz. the formation starts from two semi-pores assembled in the opposite monolayers from several molecules of the antibiotic and sterol. However, the inner diameter of the roflamycoin channel is larger because of the different antibiotic-to-sterol ratio in the channel aggregate. It is believed that the difference in the ratio is due to the presence of the methyl group in the polyene chain of roflamycoin, and the considerable difference in lifetimes of the two types of channels depends on the terminal groups of the antibiotics.     

Roflamycoin — a new channel-forming antibiotic

Ion permeability of lipid bilayers was studied in the presence of a new antifungal pentaene antibiotic, roflamycoin, the structure of which differs of which differs considerably from that of the well-known polyene channel-former amphotericin B. Both of them, however, show the property of increasing the membrane permeability only in the case of sterol-containing membrane when added on both its sides. The conductance is strongly dependent on the concentration of the antibiotic in the solutions and of sterol in the membrane. Unlike the amphotericin B channels, roflamycoin channels are potential-dependent and have short lifetime (approx. 1 s) and high conductance (approx. 100 ps in 1 M KCl), which increases linearly with the salt concentration and is not blocked by the familiar blockers of amphotericin B channels. The two antibiotics seem to have a common mechanism of channel formation, viz. the formation starts from two semi-pores assembled in the opposite monolayers from several molecules of the antibiotic and sterol. However, the inner diameter of the roflamycoin channel is larger because of the different antibiotic-to-sterol ratio in the channel aggregate. It is believed that the difference in the ratio is due to the presence of the methyl group in the polyene chain of roflamycoin, and the considerable difference in lifetimes of the two types of channels depends on the terminal groups of the antibiotics.     

New potentiators of ineffective antibiotics: Targeting the Gram-negative outer membrane to overcome intrinsic resistance

Because of the rise in antibiotic resistance and the dwindling pipeline of effective antibiotics, it is imperative to explore avenues that breathe new life into existing drugs. This is particularly important for intrinsically resistant Gram-negative bacteria, which are exceedingly difficult to treat. The Gram-negative outer membrane (OM) prevents the entry of a plethora of antibiotics that are effective against Gram-positive bacteria, despite the presence of the targets of these drugs. Uncovering molecules that increase the permeability of the OM to sensitize Gram-negative bacteria to otherwise ineffective antibiotics is an approach that has recently garnered increased attention in the field. In this review, we survey chemical matter which has been shown to potentiate antibiotics against Gram-negative bacteria by perturbing the OM. These include peptides, nanoparticles, macromolecules, antibiotic conjugates, and small molecules.     

Properties of gramicidin A channels in erythrocyte membranes

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.     

The influence of amphotericin B on the permeability of mammalian erythrocytes to nonelectrolytes, anions and cations

The influence of the polyene antibiotic, amphotericin B, on the permeability of porcine and bovine erythrocytes was studied by measuring net and tracer movements of nonelectrolytes, anions and cations in these cells.

1. 1. Amphotericin B (0.5–20 μM) enhances the rates of transfer of hydrophilic nonelectrolytes (glycerol, erythritol), anions (phosphate, lactate, glycollate, Cl⁻, SCN⁻) and cation (Na⁺, K⁺). Different concentrations of the antibiotic are required for equal effects on the different transfer processes. Bovine erythrocytes respond much less to amphotericin than porcine cells.

2. 2. Nystatin enhances the transfer of all the permeants to a much lesser extent; gramicidin D, although producing a large increase of cation permeability, leaves unaltered anion and nonelectrolyte transfer.

3. 3. The amphotericin-induced enhancement of erythrocyte permeabability (ΔP) increases with time. It has a concentration dependence of the type ΔP = α · Cⁿ_A^* (n = 1.5–2.5) and becomes more pronounced at low temperatures.

4. 4. Partial depletion of membrane cholesterol, which in itself does not alter nonelectrolyte and anion permeability, reduces the effectivity of amphotericin B, indicating that in the erythrocyte membrane, too, a sterol acts as receptor for polyene antibiotics.

5. 5. The selectivity of the amphotericin-induced pathway of transfer in the erythrocyte membrane is lower than that of the normal pathways of nonelectrolyte and anion transfer in this membrane.

The results support the view that amphotericin produces the same type of molecular reorganisation of lipid constituents in biological and artificial membranes. On the other hand, the polyene-induced pathway in the erythrocyte membrane seems to differ functionally from the normal transfer pathways in this membrane.     

One-electron reduction of an anthracycline antibiotic carminomycin by a human erythrocyte redox chain

Human erythrocyte membranes catalyse the NAD(P)H-dependent generation of the semiquinone of an adriamycin-type antibiotic carminomycin under anaerobic conditions. The maximal yield of the antibiotic radical is about 4-fold higher in the presence of NADPH than of NADH. The possible significance of the antibiotic reduction to the semiquinone by a human erythrocyte membrane redox chain for the clinical usage of these antibiotics is discussed.     

Cholesterol ester increases the permeability of erythrocyte membrane

The effect of cholesteryl palmitate on erythrocyte membrane permeability for K+ and hemoglobin was studied. Cholesterol ether was incorporated into the erythrocyte membrane by liposomes containing cholesteryl palmitate and lecithin or by dispersion of cholesteryl palmitate. It was shown that cholesteryl palmitate considerably increases permeability of the erythrocyte membrane for K+ and hemoglobin. The leakage of K+ and hemoglobin from red blood cells is not accompanied by cell destruction.     

Cerium oxide nanoparticles as potential antibiotic adjuvant. Effects of CeO2 nanoparticles on bacterial outer membrane permeability

BackgroundTherapeutic options against Multi Drug Resistant (MDR) pathogens are limited and the overall strategy would be the development of adjuvants able to enhance the activity of therapeutically available antibiotics. Non-specific outer membrane permeabilizer, like metal-oxide nanoparticles, can be used to increase the activity of antibiotics in drug-resistant pathogens. The study aims to investigate the effect of cerium oxide nanoparticles (CeO₂ NPs) on bacterial outer membrane permeability and their application in increasing the antibacterial activity of antibiotics against MDR pathogens.MethodsThe ability of CeO₂ NPs to permeabilize Gram-negative bacterial outer membrane was investigated by calcein-loaded liposomes. The extent of the damage was evaluated using lipid vesicles loaded with FITC-dextran probes. The effect on bacterial outer membrane was evaluated by measuring the coefficient of permeability at increasing concentrations of CeO₂ NPs. The interaction between CeO₂ NPs and beta-lactams was evaluated by chequerboard assay against a Klebsiella pneumoniae clinical isolate expressing high levels of resistance against those antibiotics.ResultsCalcein leakage increases as NPs concentrations increase while no leakage was observed in FITC-dextran loaded liposomes. In Escherichia coli the outer membrane permeability coefficient increases in presence of CeO₂ NPs. The antibacterial activity of beta-lactam antibiotics against K. pneumoniae was enhanced when combined with NPs.ConclusionsCeO₂ NPs increases the effectiveness of antimicrobials which activity is compromised by drug resistance mechanisms. The synergistic effect is the result of the interaction of NPs with the bacterial outer membrane. The low toxicity of CeO₂ NPs makes them attractive as antibiotic adjuvants against MDR pathogens.     

Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa.

Polycations, such as aminoglycoside and peptide antibiotics, and naturally occurring polyamines were found to bind to the lipopolysaccharide of Pseudomonas aeruginosa and alter its packing arrangement. Binding of cations was measured by the displacement of a cationic spin probe from lipopolysaccharide into the aqueous environment upon addition of competitive cations. The level of probe displacement was dependent on the concentration and charge of the competing cation, with the more highly charged cations being more effective at displacing probe. The relative affinity of several antibiotics for lipopolysaccharide correlated with their ability to increase outer membrane permeability, while the relative affinity of several polyamines correlated with their ability to stabilize the outer membrane. Probe mobility within the lipopolysaccharide head group was shown to be decreased by cationic antibiotics and unaltered or increased by polyamines. We propose that antibiotic permeability and disruption of outer membrane integrity by polycationic antibiotics results from binding of the antibiotic to anionic groups on lipopolysaccharide with a consequent change in the conformation of lipopolysaccharide aggregate structure.     

A Forward Chemical Screen Identifies Antibiotic Adjuvants in Escherichia coli

Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.     

Interactions of antibiotics of the iturin group with human erythrocytes

The peptidolipid antibiotics, iturin A and bacillomycin L have a disrupting effect on erythrocyte membrane leading to a simultaneous release of K+ ions and hemoglobin. The formation of ghosts is accompanied by a partial solubilisation of lipid components. Binding experiments with radioactive antibiotics show that about 7 X 10(7) molecules of iturin A and 4 X 10(7) molecules of bacillomycin L are bound to one erythrocyte at the concentration giving 100% hemolysis. This concentration is reduced by about 20% after treatment of erythrocytes by phospholipase A2. Scatchard plots show that the affinity for erythrocyte membrane is higher with bacillomycin L than with iturin A. This difference is probably correlated to the differences in their peptide moieties. The substitution of tyrosyl residue leads to a ten-fold increase of the concentrations giving 100% hemolysis, probably due to a low distribution coefficient of derivatives between the membrane and the solvent. This result and the humped Scatchard curves obtained with both antibiotics may be related to the self-association of the compounds in aqueous solutions.     

Multiparameter flow cytometry of bacteria: implications for diagnostics and therapeutics

BACKGROUND: Flow cytometric studies of antibiotic susceptibilities of bacteria have typically measured a single fluorescence parameter, such as membrane potential (indicating viability), or permeability to nucleic acid stains such as propidium (indicating nonviability). Cytometry of bacteria stained simultaneously with a membrane potential dye and a permeability indicator reveals unanticipated complexity. METHODS: Aliquots of cultures of three bacterial species were stained with the potential-sensitive dye hexamethylindocarbocyanine [DiIC1(3)] and the permeability indicator TO-PRO-3, in the presence and absence of a proton ionophore which collapses the potential gradient. They were analyzed using a dual-laser flow cytometer. RESULTS: Cultures grown under suboptimal conditions appear to contain cells that take up TO-PRO-3 while maintaining membrane potential, although some events showing both high DiIC1(3) fluorescence and high TO-PRO-3 fluorescence may represent clumps. CONCLUSIONS: Variations in metabolic patterns between species and within organisms under suboptimal culture conditions or following antibiotic exposure may make it difficult to develop flow cytometric clinical assays for antibiotic susceptibility. However, transient permeabilization of otherwise resistant organisms by sublethal doses of antibiotics may make it possible to treat infections by such organisms with suitably derivatized, otherwise toxic molecules; multiparameter cytometry should be useful in pursuing this approach to therapy.     

The effect of 5-(n-alk(en)yl)resorcinols on membranes. I. Characterization of the permeability increase induced by 5-(n-heptadecenyl)resorcinol

The influence of 5-(n-heptadecenyl)resorcinol, one of the main rye grain resorcinol derivatives, on the erythrocyte membrane permeability for nonelectrolytes differing in molecular size was studied turbidimetrically at various concentrations of the resorcinol derivative studied. The alkenylresorcinol induced increased permeability of the erythrocyte membrane for all the solutes studied (glycerol, m-erythritol, D-glucose, sucrose and polyethylene glycol 1000). At a given concentration of 5-(n-heptadecenyl)resorcinol the highest permeability increases were obtained for the smallest solutes. The membrane lipid to alkenylresorcinol ratio necessary for initiation of the increase of the erythrocyte membrane permeability for the solutes studied varied from 273 to 82 for glycerol and polyethylene glycol 1000, respectively, indicating that this strong membrane perturbing action may be primarily responsible for the biological effect of phenolic lipids.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Hopanoids,like sterols,modulate dynamics,compaction, phase segregation and permeability of membranes

In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.     

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.     

Characteristics of copper-induced streptomycin transport in Escherichia coli

Copper dependent uptake of streptomycin by resting E. coli cells was studied. It was shown that copper stimulates the aminoglycoside uptake only when bacteria possess endogenic energy sources. Additional accumulation of positive charged molecules of the antibiotic is accompanied by partial depolarisation of the membrane, its steady state distribution between cells and the medium corresponding to the resulted value of the membrane potential. On the basis of the data obtained it was suggested that under the influence of copper membrane permeability for streptomycin increases.     

The Properties of Ion Channels in Lipid Membranes Modified by the Aromatic Antibiotic Levorin А2

It has been shown that the main components of levorin A, that is, A₀, A₁, A₂, or A₃, that contain an aromatic group increase the permeability of membranes in the series A₃ > A₂ > A₁ > A₀ when they are on the same side of the membrane. All levorin components have cationic selectivity. The most studied levorin, А₂, promotes the almost ideal permeability of membranes to potassium ions. The membrane potential for a ten-fold change in the KCl concentration gradient is 56 ± 2 mV. It has been shown that the injection of the same concentration of levorin А₂ into one side of the membrane and then, after achieving the typical membrane permeability, into the other side of the membrane generates a two-fold increase in the total membrane permeability. This means that independent levorin-induced conductive semi-pores are formed on each side of the membrane. It has been found that the injection of levorin А₂ only into one side of the membrane enhances the membrane permeability to monosaccharides and other neutral molecules. The presence of levorin А₂ in cholesterol-, ergosterol-, and stigmasterol-containing phospholipid membranes has been shown to lead to the single-channel conductivity of typical ion channels of 0.2–0.5 pS. The properties of these channels have been studied. The levorin channels exist in two states, open and closed. Most of the time, the channel remains in the open state in the KBr solution. In solutions of different salts of the same concentration, the conductivity value of the levorin channels is approximately the same (0.4–0.5 pS). An increase in the dimethyl sulfoxide concentration in aqueous solutions facilitates the transition of polyene antibiotic molecules from dispersed to monomolecular form. The molecules of polyene antibiotics in the associated form exhibit high membrane activity.

     

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40–50% of the existing cholesterol sites.As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9–10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.     

Mechanisms of resistance to antibiotics

Microbial resistance to antibiotics is manifested by changes in antibiotic permeability, alteration of target molecules, enzymatic degradation of the antibiotics, and efflux of antimicrobials from the cytosol. Bacteria and other microorganisms use all of these mechanisms to evade the toxic effects of antibiotics. Recent research on the molecular aspects of these mechanisms, often informed by atomic resolution structures of proteins, enzymes and nucleic acids involved in these processes, has deepened our understanding of antibiotic action and resistance and, in several cases, spurred the development of strategies to overcome resistance in vitro and in vivo.