Membrane-active Antibiotics Research Articles

Interaction of Daptomycin and Calcium with Lipid Bilayers

Daptomycin is a cyclic lipopeptide antibiotic recently approved for clinical treatment of gram positive bacterial infections. It displays strong bactericidal behavior in the presence of excess calcium ions and in biomembranes containing negatively charged PG headgroups. Yet, the molecular mechanism of daptomycin, beyond the suggestion of ion channel formation and cellular depolarization, remains unknown. Recent data has shown results inconsistent with ion channel formation, prompting a more detailed investigation of Daptomycin and calcium interactions with lipid bilayers at higher structural resolutions. In order to fully characterize the mechanism of Daptomycin, it is necessary to understand the role of individual components that are integral to Daptomycin's mode of action in the lipid bilayer. By using lamellar diffraction of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) multilayers, we examined the individual and combined effects of Daptomycin and calcium ions on bilayer structure. We found that calcium ions alone result in a slight thickening effect of the bilayer, partially supporting molecular simulations of calcium ions and PG bilayers that propose a calcium ordering effect at physiological calcium concentrations. Inclusion of daptomycin alone produced a bilayer thinning effect, suggesting that Daptomycin is capable of inserting into the headgroup region of the lipid bilayer during binding. Lastly, samples that contained both Daptomycin and calcium ions showed a bilayer thickening effect. These results suggest that Daptomycin binds to the membrane in the absence of calcium ions but induces a structural thickening of the bilayer in the presence of calcium ions. The apparent thickening effect of Daptomycin is unlike other membrane-active antibiotics, such as melittin or LL-37, which induce bilayer thinning. These studies may form the basis for more detailed investigations to characterize the molecular mechanisms of Daptomycin and other cyclic lipopeptides.

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains

Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.

Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics.

Persisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active against Escherichia coli persisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect on E. coli persisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugs in vitro The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

Could targeted, antibiotic-loaded gold nanoconstructs be a new magic bullet to fight infection?

Could targeted, antibiotic-loaded gold nanoconstructs be a new magic bullet to fight infection?

Self-enhanced targeted delivery of a cell wall– and membrane-active antibiotics, daptomycin, against staphylococcal pneumonia

Considering that some antibacterial agents can identify the outer structure of pathogens like cell wall and/or cell membrane, we explored a self-enhanced targeted delivery strategy by which a small amount of the antibiotic molecules were modified on the surface of carriers as targeting ligands of certain bacteria while more antibiotic molecules were loaded inside the carriers, and thus has the potential to improve the drug concentration at the infection site, enhance efficacy and reduce potential toxicity. In this study, a novel targeted delivery system against methicillin-resistant Staphylococcus aureus (MRSA) pneumonia was constructed with daptomycin, a lipopeptide antibiotic, which can bind to the cell wall of S. aureus via its hydrophobic tail. Daptomycin was conjugated with N-hydroxysuccinimidyl–polyethylene glycol–1,2-distearoyl-sn-glycero-3-phosphoethanolamine to synthesize a targeting compound (Dapt–PEG–DSPE) which could be anchored on the surface of liposomes, while additional daptomycin molecules were encapsulated inside the liposomes. These daptomycin-modified, daptomycin-loaded liposomes (DPD-L[D]) showed specific binding to MRSA as detected by flow cytometry and good targeting capabilities in vivo to MRSA-infected lungs in a pneumonia model. DPD-L[D] exhibited more favorable antibacterial efficacy against MRSA than conventional PEGylated liposomal daptomycin both in vitro and in vivo. Our study demonstrates that daptomycin-modified liposomes can enhance MRSA-targeted delivery of encapsulated antibiotic, suggesting a novel drug delivery approach for existing antimicrobial agents.

Membrane Sterols Modulate the Binding Mode of Amphotericin B without Affecting Its Affinity for a Lipid Bilayer.

Membrane-active antibiotics are known to selectively target certain pathogens based on cell membrane properties, such as fluidity, lipid ordering, and phase behavior. These are in turn modulated by the composition of a lipid bilayer and in particular by the presence and type of membrane sterols. Amphotericin B (AmB), the golden standard of antifungal treatment, exhibits higher activity toward ergosterol-rich fungal membranes, which permits its use against systemic mycoses; however, the selectivity for fungal membranes is far from satisfactory leading to severe side effects. Despite decades of research, no consensus has emerged on the origin of AmB specificity for fungal cells and its actual mode of action at the molecular level. Previously, it has been proposed that the specific action of AmB is related to differences in its affinity for membranes of different composition. In this work, we investigate this relationship by employing molecular dynamics simulations to compare the free energy of insertion of AmB into three types of membranes: a pure DMPC bilayer and DMPC bilayers containing 30% of cholesterol or ergosterol. We analyze the orientation of AmB molecules within the bilayer in order to unambiguously establish their membrane binding mode and relate the orientational freedom to the sterol-dependent tightness of lipid packing. Our results strongly indicate that the membrane insertion of AmB proceeds virtually to completion independent of membrane type, and hence the higher toxicity against fungal membranes may rather result from differences in subsequent oligomerization in the membrane and assembly of monomers into functional transmembrane pores. In particular, the latter could be facilitated by sterol-induced ordering of AmB molecules along the membrane normal, revealed by our free energy profiles. Moreover--in contrast to certain claims--we find no stable binding mode corresponding to the horizontal adsorption of AmB on the membrane surface.

Nanostructure-Dependent Antimicrobiotic Activity and Selectivity of Synthetic Membrane-Active Polymers

Antibiotic resistance has become one of the greatest challenges in treating bacterial infections in healthcare. Inspired by the structures of bacteria-invading viruses and antimicrobial peptides (AMPs), we hypothesize that in addition to a balance of amphiphilicity and electropositivity, nanostructure is another important structural determinant that defines how membrane-active antibiotics remodel host membranes to gain desirable activity and selectivity. Here we study the structure-activity relationship of a series of polymer molecular brushes (PMBs) with well-defined nanostructures that mimic spherical and rod-shaped viruses. Our preliminary data based on PMBs with hydrophilic polymer brushes reveal that: (1) amphiphilicity is not a required trait - hydrophilic PMBs can be designed to have potent antibiotic performance as well with negligible hemolytic activity; (2) the nanoscale architecture of PMBs defines their double selectivity, not molecular weight per se; (3) PMBs are far more powerful antibiotics than individual linear-chain polymers that make up the PMBs; and (4) nanostructured PMBs induce topological changes of membranes by forming membrane pores that unlikely fit in with any known models of AMP action. These findings expand existing wisdom on designing synthetic mimics of AMPs and suggest that the spatially-defined, multivalent interactions inherent to nanostructured PMBs is of great significance for the development of new membrane-active antibiotics.

Intracellular activity of a membrane-active glycopeptide antibiotic against meticillin-resistant Staphylococcus aureus infection.

Staphylococcus aureus is a facultative intracellular pathogen and there are limited options for the treatment of severe intracellular bacterial infections. The membrane-active glycopeptide antibiotic Van-QC8 is a permanent positively charged lipophilic vancomycin analogue that demonstrates high activity against clinically relevant drug-resistant Gram-positive bacteria both in vitro and in vivo. In this study, the intracellular activity of Van-QC8 was evaluated against meticillin-resistant S. aureus (MRSA) infection in RAW macrophages. Furthermore, the mechanism of intracellular uptake of Van-QC8 was investigated. Van-QC8 showed time- and concentration-dependent bactericidal activity against intracellular MRSA. Van-QC8 displayed significantly higher intracellular activity compared with vancomycin and linezolid. Cellular uptake of Van-QC8 was found to be through clathrin-dependent and -independent and caveolin-dependent and -independent endocytic pathways. The findings of this study suggest that Van-QC8 could be translated clinically for the treatment of intracellular infections due to MRSA.

Protective role of E. coli outer membrane vesicles against antibiotics

The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics.

Mode of action and bactericidal properties of surotomycin against growing and nongrowing Clostridium difficile.

Surotomycin (CB-183,315), a cyclic lipopeptide, is in phase 3 clinical development for the treatment of Clostridium difficile infection. We report here the further characterization of the in vitro mode of action of surotomycin, including its activity against growing and nongrowing C. difficile. This was assessed through time-kill kinetics, allowing a determination of the effects on the membrane potential and permeability and macromolecular synthesis in C. difficile. Against representative strains of C. difficile, surotomycin displayed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures at a concentration that was ≤16× the MIC. Exposure resulted in the inhibition of macromolecular synthesis (in DNA, RNA, proteins, and cell wall). At bactericidal concentrations, surotomycin dissipated the membrane potential of C. difficile without changes to the permeability of propidium iodide. These observations are consistent with surotomycin acting as a membrane-active antibiotic, exhibiting rapid bactericidal activities against growing and nongrowing C. difficile.

Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells

Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the United States. When treated early, the disease usually resolves, but when left untreated, it can result in symptoms such as arthritis and encephalopathy. Treatment of the late-stage disease may require multiple courses of antibiotic therapy. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late-stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In this study, we examined the ability of B. burgdorferi to form persisters. Killing growing cultures of B. burgdorferi with antibiotics used to treat the disease was distinctly biphasic, with a small subpopulation of surviving cells. Upon regrowth, these cells formed a new subpopulation of antibiotic-tolerant cells, indicating that these are persisters rather than resistant mutants. The level of persisters increased sharply as the culture transitioned from the exponential to stationary phase. Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary-phase cells but not persisters. Mitomycin C, an anticancer agent that forms adducts with DNA, killed persisters and eradicated growing and stationary cultures of B. burgdorferi. Finally, we examined the ability of pulse dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and the antibiotic was added again. Four pulse doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture.

Virus-Mimicking Polymer Molecular Brushes Are Potent Antibiotics with Double Selectivity

Evolution of antibiotics-resisting pathogens has become one of the greatest challenges in the battle of bacterial infection. Inspired by the structures of bacteria-invading viruses and antimicrobial peptides, we hypothesize that in addition to a balance of amphiphilicity and electropositivity, the nanoscale architecture is an essential determinant that dictates how membrane-active antibiotics remodel host membranes to achieve desirable activity and selectivity. Here we study the structure-activity relationship of a series of polymer molecular brushes (PMBs) with well-defined nanoscale architectures that mimic spherical and rod-shaped viruses. Our preliminary data based on PMBs with hydrophilic polymer brushes reveal that: (1) amphiphilicity is not a required trait - hydrophilic PMBs can be designed to have potent antibiotic performance as well with no hemolytic side effect; (2) the nanoscale architecture of PMBs defines their double selectivity, not molecular weight per se; (3) PMBs are far more powerful antibiotics than individual linear-chain polymers that make up the PMBs; and (4) nanostructured PMBs induce topological changes of membranes by forming membrane pores that unlikely fit in with any known models of AMP action. These findings challenge existing wisdom and suggest that the spatially-defined, multivalent interactions inherent to PMBs is of great significance for the development of polymer-based antibiotics.

In vitro and in vivo safety and efficacy studies of amphotericin B on Babesia gibsoni

Babesia gibsoni is a causative pathogen of canine babesiosis, which is commonly treated with anti-babesial drugs; however, the development of novel, more effective anti-babesial drugs is necessary because the currently used drugs cannot remove the parasites from dogs. Therefore we investigated the anti-babesial effect of amphotericin B (AmB), a membrane-active polyene macrolide antibiotic. The interaction of such compounds with sterols in bilayer cell membranes can lead to cell damage and ultimately cell lysis. AmB exhibits in vitro activity against B. gibsoni in normal canine erythrocytes within 12h. We also studied liposomal AmB (L-AmB), a liposomal formulation of AmB that required a longer incubation period to reduce the number of parasites. However, L-AmB completely inhibited the invasion of free parasites into erythrocytes. These results indicated that free parasites failed to invade erythrocytes in the presence of L-AmB. Both AmB and L-AmB induced mild hemolysis of erythrocytes. Moreover, the methemoglobin level and the turbidity index of erythrocytes were significantly increased when erythrocytes were incubated with AmB, suggesting that AmB induced oxidative damage in erythrocytes. Finally, the anti-babesial activity of AmB in vivo was observed. When experimentally B. gibsoni-infected dogs were administered 0.5 and 1mg/kg AmB by the intravenous route, the number of parasites decreased; however, recurrence of parasitemia was observed, indicating that AmB did not eliminate parasites completely. Blood urea nitrogen and creatinine of dogs were abnormally elevated after the administration of 1mg/kg AmB. These results indicate that AmB has in vivo activity against B. gibsoni; however, it does not eliminate parasites from infected dogs and affects kidney function at a high dose.

Chemical Modulation of the Biological Activity of Reutericyclin: a Membrane-Active Antibiotic from Lactobacillusreuteri

Whilst the development of membrane-active antibiotics is now an attractive therapeutic concept, progress in this area is disadvantaged by poor knowledge of the structure-activity relationship (SAR) required for optimizing molecules to selectively target bacteria. This prompted us to explore the SAR of the Lactobacillus reuteri membrane-active antibiotic reutericyclin, modifying three key positions about its tetramic acid core. The SAR revealed that lipophilic analogs were generally more active against Gram-positive pathogens, but introduction of polar and charged substituents diminished their activity. This was confirmed by cytometric assays showing that inactive compounds failed to dissipate the membrane potential. Radiolabeled substrate assays indicated that dissipation of the membrane potential by active reutericyclins correlated with inhibition of macromolecular synthesis in cells. However, compounds with good antibacterial activities also showed cytotoxicity against Vero cells and hemolytic activity. Although this study highlights the challenge of optimizing membrane-active antibiotics, it shows that by increasing antibacterial potency the selectivity index could be widened, allowing use of lower non-cytotoxic doses.

Molecular Characterization and Functional Analysis of Outer Membrane Vesicles from the Antarctic Bacterium Pseudomonas syringae Suggest a Possible Response to Environmental Conditions

Outer membrane vesicles (OMVs) of Gram-negative bacteria form an important aspect of bacterial physiology as they are involved in various functions essential for their survival. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz4W were isolated, and the proteins and lipids they contain were identified. The matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) analysis revealed that phosphatidylethanolamines and phosphatidylglycerols are the main lipid components. The proteins of these vesicles were identified by separating them by one-dimensional gel electrophoresis and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). They are composed of outer membrane and periplasmic proteins according to the subcellular localization predictions by Psortb v.3 and Cello V2.5. The functional annotation and gene ontology of these proteins provided hints for various functions attributed to OMVs and suggested a potential mechanism to respond to the extracellular environmental changes. The OMVs were found to protect the producer organism against the membrane active antibiotics colistin and melittin but not from streptomycin. The 1-N-phenylnapthylamine (NPN)-uptake assay revealed that the OMVs protect the bacterium from membrane active antibiotics by scavenging them and also showed that membrane and protein packing of the OMVs was similar to the parent bacterium. The sequestering depends on the composition and organization of lipids and proteins in the OMVs.

Using Spheroplasts to Study Peptide Interactions with Cell Membranes

Cytoplasmic membranes remain intact if bacteria cells are stripped off the outer cell wall and the peptidoglycan-they are called spheroplasts or protoplasts. This allows us to study the interaction of membrane-active antibiotics directly with the cytoplasmic membranes of bacterial. For our purpose, we want giant spheroplasts in order to apply the aspiration techniques. The technique of aspiration serves two purposes: one is to apply tension to the membrane and another is to measure the membrane area changes. The measurement of the membrane area change by tension or peptide binding or any structural event is the best physical quantitative description for the state of the membrane. We believe that these experiments will lead to discover the so-far unknown properties of cell membranes. Using established methods we grew spheroplasts which were stabilized in a STOP solution. To test the condition of the cytoplasmic membranes, we then diluted the STOP solution by adding pure water. We found that as the STOP solution was diluted, the water influx enlarged the spheroplasts. This seems to indicate that there is a membrane reservoir (perhaps, invaginations of cell membrane) in the original state of spheroplast. By the aspiration method, we measured the tension versus membrane area change of spheroplasts stabilized at 15% STOP solution. The tension vs. area change showed a slow exponential region followed by a linear region, similar to pure lipid GUVs. However the stretch expansion moduli are 5 times smaller than that of pure lipid bilayers. When maganin or melittin, were introduced into spheroplast suspension, we found that the surface area was expanded by more than 5% by peptide binding. These preliminary results indicate the feasibility of using spheroplasts as an experimental platform for studying the interaction of membrane-active peptides with cell membranes.

Peptaibol Antiamoebin I: Spatial Structure, Backbone Dynamics, Interaction with Bicelles and Lipid‐Protein Nanodiscs, and Pore Formation in Context of Barrel‐Stave Model

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal (1-8) region. We studied Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles)(1) ) by heteronuclear (1) H,(13) C,(15) N-NMR spectroscopy. Interaction with the bicelles stabilizes the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-μs time scale. Extensive ms-μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv-IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs-ms time scale. Titration with Mn(2+) and 16-doxylstearate paramagnetic probes revealed Aam-I binding to the bicelle surface with the N-terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam-I helix between surface-bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short-chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel-stave model of TM pore formation, similarly to the mechanism proposed for Zrv-IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam-I.

Design and Synthesis of Amphiphilic Xanthone-Based, Membrane-Targeting Antimicrobials with Improved Membrane Selectivity

This work describes how to tune the amphiphilic conformation of α-mangostin, a natural compound that contains a hydrophobic xanthone scaffold, to improve its antimicrobial activity and selectivity for Gram-positive bacteria. A series of xanthone derivatives was obtained by cationic modification of the free C3 and C6 hydroxyl groups of α-mangostin with amine groups of different pKa values. Modified structures using moieties with high pKa values, such as AM-0016 (3b), exhibited potent antimicrobial properties against Gram-positive bacteria. Compound 3b also killed bacteria rapidly without inducing drug resistance and was nontoxic when applied topically. Biophysical studies and molecular dynamics simulations revealed that 3b targets the bacterial inner membrane, forming an amphiphilic conformation at the hydrophobic-water interface. In contrast, moieties with low pKa values reduced the antimicrobial activity of the parent compound when conjugated to the xanthone scaffold. This strategy provides a new way to improve "hits" for the development of membrane-active antibiotics that target drug-resistant pathogens.

Exploring Amphotericin B-Membrane Interactions: Free Energy Simulations

Amphotericin B (AmB) is a membrane-active polyene antibiotic used to treat serious fungal infections. Biological action of AmB is due to the formation of transmembrane channels. Membrane sterols are known to be crucial for the AmB antifungal activity, i.e. AmB is more active against fungal cell membranes containing ergosterol than against the mammalian membranes with cholesterol.We explored molecular determinants of AmB selectivity for ergosterol-containing membranes using computational methods. By means of molecular dynamics simulations we studied various aspects of the interactions between AmB and lipid bilayers of different composition (containing or not 30% of ergosterol or cholesterol). More precisely, we examined (1) AmB insertion into a membrane, (2) changing tilt angle between AmB and the bilayer plane (for AmB embedded in a membrane) as well as (3) AmB dimerization in a membrane. To provide a thermodynamic description of these processes, we calculated the free energy profiles describing each of them in the three different membrane systems. The results indicate that at low, chemotherapeutically relevant concentrations of AmB at which the antibiotic expresses its channel-forming activity, AmB is mostly monomeric in ergosterol-containing membranes and it exists predominantly as a dimer in cholesterol-containing (and sterol-free) ones. We also show that compared to the other two studied membranes, it is the most favorable for AmB to insert the ergosterol-containing bilayer. The differences in the behavior patterns of AmB in bilayers of different composition are mainly of energetic origin. From the free energy profiles for (2) and (3) we also determine the most preferred location and orientation of AmB within the studied bilayers.

Selective antimicrobial activity and mode of action of adepantins, glycine-rich peptide antibiotics based on anuran antimicrobial peptide sequences

A challenge when designing membrane-active peptide antibiotics with therapeutic potential is how to ensure a useful antibacterial activity whilst avoiding unacceptable cytotoxicity for host cells. Understanding their mode of interaction with membranes and the reasons underlying their ability to distinguish between bacterial and eukaryotic cytoplasmic cells is crucial for any rational attempt to improve this selectivity. We have approached this problem by analysing natural helical antimicrobial peptides of anuran origin, using a structure–activity database to determine an antimicrobial selectivity index (SI) relating the minimal inhibitory concentration against Escherichia coli to the haemolytic activity (SI=HC50/MIC). A parameter that correlated strongly with SI, derived from the lengthwise asymmetry of the peptides' hydrophobicity (sequence moment), was then used in the “Designer” algorithm to propose novel, highly selective peptides. Amongst these are the ‘adepantins’, peptides rich in glycines and lysines that are highly selective for Gram-negative bacteria, have an exceptionally low haemolytic activity, and are less than 50% homologous to any other natural or synthetic antimicrobial peptide. In particular, they showed a very high SI for E. coli (up to 400) whilst maintaining an antimicrobial activity in the 0.5–4μM range. Experiments with monomeric, dimeric and fluorescently labelled versions of the adepantins, using different bacterial strains, host cells and model membrane systems provided insight into their mechanism of action.