Antimicrobial Lipopeptides Research Articles

Estimating the Free Energy to Bind a Potent Antimicrobial Lipopeptide to a Model Membrane Bilayer

A series of synthetic antimicrobial lipopeptides (AMLPs) based around a common architecture of 4 amino acids (2 lysines), with a saturated fatty acid conjugated to the N-terminus, have been shown to have broad-spectrum antimicrobial activity and low hemolytic activity. Previous all-atom and coarse-grained molecular dynamics simulations from our group have shown that these molecules form micelles in solution and readily bind to model lipid bilayers. Here, we used microsecond-scale coarse-grained molecular dynamic simulations with the MARTINI forcefield to explore the thermodynamics governing the binding process, considering both isolated lipopeptides molecules and the micellar state.

Non-leaching antimicrobial surfaces through polydopamine bio-inspired coating of quaternary ammonium salts or an ultrashort antimicrobial lipopeptide

Bacterial fouling on surfaces significantly increases the resistance of bacteria toward antibiotics, which leads to medical complications and a corresponding financial burden. Here, we report on a general and robust technique for facile modification of various surfaces with different antibacterial agents. Our approach in this study was inspired by the strong adhesion of mussel adhesion proteins (MAPs) to many types of surfaces, including metals, polymers, and inorganic materials. Thus, glass and polymeric slides were dip-coated with dopamine, as a MAP mimic, and the resulting surfaces were characterized. The reactivity of dopamine-coated surfaces toward nucleophilic addition was then confirmed by reacting them with fluorescent probes containing either a free amino or a free thiol group. Laser scanning confocal microscopy (LSCM), X-ray photoelectron spectroscopy (XPS), confocal Raman microscopy, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy, and cyclic voltammetry studies collectively suggested that the probes had covalently attached to the surfaces. Fabrication of dopamine-coated surfaces with an antibacterial quaternary amine or an ultrashort lipopeptide analog generated surfaces that effectively kill Escherichia coli and Staphylococcus aureuscells on contact. Moreover, minimal leaching of the fabricated agent was detected after prolonged incubation. This technique could be further developed to a “paint-like” or self-assembling monolayer-like procedure for the preparation of antibacterial surfaces on various materials.

Functions of antimicrobial lipopeptides produced by antagonistic Bacillus spp. in suppression for plant diseases

Functions of antimicrobial lipopeptides produced by antagonistic Bacillus spp. in suppression for plant diseases

Characterization of a potent antimicrobial lipopeptide via coarse-grained molecular dynamics

The prevalence of antibiotic-resistant pathogens is a major medical concern, prompting increased interest in the development of novel antimicrobial compounds. One such set of naturally occurring compounds, known as antimicrobial peptides (AMPs), have broad-spectrum activity, but come with many limitations for clinical use. Recent work has resulted in a set of antimicrobial lipopeptides (AMLPs) with micromolar minimum inhibitory concentrations and excellent selectivity for bacterial membranes. To characterize a potent, synthetic lipopeptide, C16-KGG K, we used multi-microsecond coarse-grained simulations with the MARTINI forcefield, with a total simulation time of nearly 46 μs. These simulations show rapid binding of C16-KGG K, which forms micelles in solution, to model bacterial lipid bilayers. Furthermore, upon binding to the surface of the bilayer, these lipopeptides alter the local lipid organization by recruiting negatively charged POPG lipids to the site of binding. It is likely that this drastic reorganization of the bilayer has major effects on bilayer dynamics and cellular processes that depend on specific bilayer compositions. By contrast, the simulations revealed no association between the lipopeptides and model mammalian bilayers. These simulations provide biophysical insights into lipopeptide selectivity and suggest a possible mechanism for antimicrobial action. This article is part of a Special Issue entitled: Membrane protein structure and function.

Specific Interactions of Mycosubtilin with Cholesterol-Containing Artificial Membranes

Mycosubtilin is a natural antimicrobial lipopeptide produced by Bacillus subtilis strains. It is characterized by its hemolytic and strong antifungal activities. Mycosubtilin interacts with the plasma membranes of sensitive cells. However, the molecular mechanisms of its biological activities have not been completely elucidated. Our purpose was therefore to analyze the interactions of mycosubtilin with biological membranes by using biomimetic membranes such as Langmuir monolayers and multilayers. Structural changes of mycosubtilin, involving its peptide backbone and the side chain of its tyrosyl residue, were observed when the lipopeptide was interacting with cholesterol-containing multilayers. The interactions of mycosubtilin with monolayers constituted by pure lipids and by phosholipid/cholesterol or phospholipid/sphingomyelin/cholesterol were also examined. An original behavior of mycosubtilin toward cholesterol-containing monolayers was found. However, this original behavior was lost when mycosubtilin was interacting with pure cholesterylacetate monolayers. This suggests the involvement of the alcohol group of cholesterol in mycosubtilin-cholesterol interactions within membranes. Moreover, mycosubtilin induced changes in the organization and morphology of cholesterol-containing monolayers, and large condensed domains with different levels of condensation appeared only in the case of DPPC/sphingomyelin/cholesterol monolayer.

A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides

A modified electroporation method using trehalose is presented for the transformation of Bacillus subtilis. The new method improved the transformation efficiency of B. subtilis nearly 2,000-fold compared with the usual method, giving 4×10(5) transformants/μg DNA. Using this method, B. subtilis was engineered to improve production of antimicrobial lipopeptides and produced 1.8-fold more surfactin and 2.9-fold more fengycin.

Diversity and applications ofBacillusbacteriocins

Members of the genus Bacillus are known to produce a wide arsenal of antimicrobial substances, including peptide and lipopeptide antibiotics, and bacteriocins. Many of the Bacillus bacteriocins belong to the lantibiotics, a category of post-translationally modified peptides widely disseminated among different bacterial clades. Lantibiotics are among the best-characterized antimicrobial peptides at the levels of peptide structure, genetic determinants and biosynthesis mechanisms. Members of the genus Bacillus also produce many other nonmodified bacteriocins, some of which resemble the pediocin-like bacteriocins of the lactic acid bacteria (LAB), while others show completely novel peptide sequences. Bacillus bacteriocins are increasingly becoming more important due to their sometimes broader spectra of inhibition (as compared with most LAB bacteriocins), which may include Gram-negative bacteria, yeasts or fungi, in addition to Gram-positive species, some of which are known to be pathogenic to humans and/or animals. The present review provides a general overview of Bacillus bacteriocins, including primary structure, biochemical and genetic characterization, classification and potential applications in food preservation as natural preservatives and in human and animal health as alternatives to conventional antibiotics. Furthermore, it addresses their environmental applications, such as bioprotection against the pre- and post-harvest decay of vegetables, or as plant growth promoters.

Short Alkylated Peptoid Mimics of Antimicrobial Lipopeptides

We report the creation of alkylated poly-N-substituted glycine (peptoid) mimics of antimicrobial lipopeptides with alkyl tails ranging from 5 to 13 carbons. In several cases, alkylation significantly improved the selectivity of the peptoids with no loss in antimicrobial potency. Using this technique, we synthesized an antimicrobial peptoid only 5 monomers in length with selective, broad-spectrum antimicrobial activity as potent as previously reported dodecameric peptoids and the antimicrobial peptide pexiganan.

Antimicrobial Activity of Short, Synthetic Cationic Lipopeptides

The increasing emergence of multidrug-resistant micro-organisms presents one of the greatest challenges in the clinical management of infectious diseases. Therefore, novel antimicrobial agents are urgently required to address this issue. In this report, we describe the solid phase synthesis, characterization, microbiological and toxicological evaluation of a library of ultrashort cationic antimicrobial lipopeptides based on the previously described tetrapeptide amide H-Orn-Orn-Trp-Trp-NH2 conjugated with saturated fatty acids which have inherent antimicrobial activity. The microbiological activity of these ultrashort cationic lipopeptides, which exhibit excellent, broad-spectrum antimicrobial activity against a number of clinically important pathogenic bacteria and fungi, including multidrug resistant micro-organisms in both planktonic and sessile (biofilm) cultures is reported.

Separation and extraction of antimicrobial lipopeptides produced by Bacillus amyloliquefaciens ES-2 with macroporous resin

A new method for separation and extraction of antibiotic lipopeptides produced by Bacillus amyloliquefaciens ES-2 with macroporous resin was carried out in this study. Among widely used twelve macroporous resins, X-5 resin had the highest adsorption capacity in static and dynamic tests, its non-polarity and configuration were suited for the lipopeptides (surfactin and fengycin). Dynamic adsorption and desorption experiments were performed using a column packed with X-5 resin to obtain optimal conditions, with 30 g/L resin concentration, 0.5 BV/h feed flow rate, 80% aqueous ethanol at 1 BV/h elute flow rate as eluant agent, the total content of lipopeptides in final product was 79.5% (surfactin content was 33.2% while fengycin content was 23.4%), which had been improved about 150% compared with that of lipopeptide in fermentation broth. Because of its low cost, high efficiency, procedural simplicity and scale up ease, the adsorption–desorption method could be more efficient for the preparative separation and extraction of antibiotic lipopeptide than the conventional acid precipitation method, which could provide scientific references for antibiotic lipopeptide production.

Novel drug delivery system for lipophilic therapeutics of small molecule, peptide‐based and protein drugs

Effective drug delivery systems for biopharmaceuticals, such as proteins, peptide-based drugs, and vaccines still present major challenges. A procedure has been developed whereby human serum albumin (HSA) can be used as a delivery vehicle for these biomolecules exploiting its role as main fatty acid carrier. Using essentially fatty acid free HSA (HSAff) it is possible to form stable complexes with lipidic chain compounds (lipo compounds). Several lipo compounds have been used to develop this system, a novel antimicrobial lipopeptide and γ-amino-n-butyric acid, GABA, conjugated with an alkyl chain, lipo-GABA, in both cases C8 and C14 alkyl chain lengths were evaluated. The HSA-lipo complex had a mutual stabilizing effect on both the HSA and the lipo compound. The protease enzyme study showed that the alkyl chains of these lipo compounds bound to HSAff confer biostability to HSAff. With the antimicrobial lipopeptide, there was enhanced activity with HSAff formulation suggesting increased biostability and bioavailability of compounds. These finding allowed us to propose a simple and effective way of delivering lipo compounds using fatty acid free HSA as the carrier.

Nanostructure Determines Antifungal Activity of De Novo Designed pH Dependent Histidine Containing Ultra-Short Lipopeptides

Antimicrobial peptides are an essential part of the innate immune system of most living things and the understanding of the biophysical properties and the different mechanisms of action are crucial for the de-novo development of simple and effective analogs. More specifically, antimicrobial lipopeptides have been gaining increased attention because of the pressure for new antimicrobial agents against resistant pathogens. The addition of a lipophilic fatty acid has proven to be an effective method to increase the association of a peptide with the membrane, thus increasing the biological activity of certain peptide sequences. Previously, we reported that linear ultrashort cationic lipopeptides even as short as 4 amino acids have potent antimicrobial and antifungal properties. We described the minimum peptide length, and fatty acid length necessary for activity. Also, innate-immunity-like peptides were described that contained multiple histidine residues. Although these peptides consisted of 12 to 15 amino acids, these were less toxic to the host, and were lytic to numerous pathogens and cancer cells at slightly acidic environments. Here we report the design of an ultrashort histidine containing peptide whose antifungal activity could be significantly increased in a covalent trimeric form. Low micromolar activity was observed for Aspergillus fumigatus and Cryptococcus neoformans but not Candida albecans. Using transmission electron microscopy, we observed that this trimeric ultrashort histidine containing peptide formed distinct and differing nanostructures at pH 5 and 7, which could explain the activity differences. Since various organs or areas of the human body have a slightly acidic pH environment such as tumors, gastric lumen and lung-lining fluids in cystic fibrosis and asthma, understanding the importance of nanostructure-activity relationships of these pH dependent ultrashort peptides could lead to improvements in the delivery and administration of the peptides.

Binding of Antimicrobial Lipopeptides To Lipid Bilayers Characterized By Microsecond Molecular Dynamics Simulations

The emergence of antibiotic resistant pathogens is one of the major medical problems of the 21st century, prompting renewed interest in the development of novel antimicrobial compounds. Here we use microsecond-scale all-atom molecular dynamics simulations to characterize the structure, dynamics, and membrane-binding mechanism of a synthetic antimicrobial lipopeptide, C16-KGGK. The results of the simulations are validated by comparison with solid state NMR experiments, and yield new insights into the molecules' mechanism of action.

Effect of Dipole Modifying Agents on the Surfactin Induced Conductance of Planar Lipid Bilayers

We studied effects of compounds changing membrane dipole potential on membrane conductance induced by antimicrobial lipopeptide surfactin. Surfactin added on both sides of an artificial lipid bilayer from diphytanoyl phosphocholine in 1 M KCl (pH 6.5) produces an increase of the membrane conductance as a result of ion channel formation. Increasing a membrane dipole potential adding RH 421 to the bilayer bathing solution (10 µM , both sides) leads to ∼40 times increase of a steady-state conductance. At the same time, addition of phloretin (20 µM), known to decrease the dipole potential, results in decrease of the surfactin-induced conductance by ∼30 times. We note, that the effects of dipole modifiers on the surfactin-induced membrane conductance are clearly opposite to the effects observed with the same modifiers in case of syringomycin E-induced conductance of lipid bilayers [Ostroumova et al., Langmuir, 2008]. As we suggested earlier, the influence of dipole modifiers on syringomycin activity may be related to a promotion/retardation of a movement of positively charged syringomycin molecules in the direction of membrane hydrocarbon core. In contrast to syringomycin, surfactin is negatively charged. Hence, one can expect an inversion of the effect of dipole modifiers in case of surfactin. The obtained results are in agreement with the model proposed in [Ostroumova et al., Langmuir, 2008].The study was supported in part by the Russian Fund for Basic Research (09-04-48860), the State Program of Molecular and Cell Biology, the grant of St.-Petersburg Administration for young scientists, and the State contract (FAE Π-1372).

Antimicrobial Lipopeptides In Anionic And Zwitterionic Membranes Investigated By Molecules Dynamics Simulations

The explosive development of new antibiotic resistant strains of bacteria makes it essential for us to develop new antibiotic drugs, preferably one which operate via novel pathways. Compounds based on antimicrobial peptides seem like good candidates, because they are broadly effective and largely resilient to evolved immunity. However, natural peptides tend to be much larger than typical drugs, expensive to synthesize, and tend to be rapidly digested in the bloodstream. Shai and coworkers have recently begun invesitgating a new set of compounds, synthetic antimicrobial lipopeptides, designed to have many of the strengths of natural peptides while avoiding many of their weaknesses. Here we use all-atom molecular dynamics of simulations of two of the peptides they used -- C16-KGGK and C16-KGGK -- bound to zwitterionic, “mammlian-like” membranes (POPC) and anionic, “bacterial-like” membranes (POPE:POPG). The variations in their structure and dynamics suggest new insights into the mechanism of selectivity and function.

New Trends in Pharmacogenomic Strategies Against Resistance Development in Microbial Infections

This review summarizes some of the new trends in the fight against drug resistant bacteria. We review Gram-positive (e.g., S.aureus) and Gram-negative (e.g., Pseudomonas aeruginosa, Helicobacter pylori) bacteria, the current antibiotic resistance situation, as well as resistance spread and some recently discovered resistance mechanisms, such as those based on integrons and complex transposons. We then summarize several current routes to identify new drugs such as cationic antimicrobial peptides, novel acyldepsipeptides, RNA aptamers and lipopeptides. New drug strategies to treat resistant pathogens include eliciting growth in dormant bacteria, or a new way to attack efflux systems. Typical approaches from pharmacogenomics combined with systems biology and bioinformatics support these routes (simulations, metagenomics and metabolic network modeling), as well as the patient treatment (e.g., haplotyping and immune response).

Antimicrobial Lipopolypeptides Composed of Palmitoyl Di- and Tricationic Peptides: In Vitro and in Vivo Activities, Self-Assembly to Nanostructures, and a Plausible Mode of Action

Antimicrobial lipopeptides are produced nonribosomally in bacteria and fungi during cultivation. They are composed of a cationic or an anionic peptide covalently bound to a specifically modified aliphatic chain. Most of the peptidic moieties have complex cyclic structures. Here we report that conjugation of a palmitic acid to the N-terminus of very short cationic di- and tripeptides composed of all l- and d, l-amino acids endowed them with potent antimicrobial activities. Interestingly, cell specificity was determined by the sequence of the short peptidic chain. Palmitoyllysine served as a control and was inactive toward all microorganisms tested. Replacing an l-amino acid with its d-enantiomer did not affect the activity of the corresponding lipopeptides. Importantly, selected lipopeptides were also potent in vivo in a mouse model of Candida albicans infection. Bacterial leakage experiments and negative staining electron microscopy suggest that their mode of action involves permeation and disintegration of the microorganism's membrane, similar to many long antimicrobial peptides and lipopeptides. Interestingly, each lipopeptide assembled in solution into a nanostructure with a unique morphology which could partially explain differences in their biological activity. Besides adding important information on the parameters necessary for antimicrobial lipopeptides to kill microorganisms, the simple composition of these minilipopeptides and their diverse cell specificities make them attractive candidates for various applications.

Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans

To isolate the biologically active fraction of the lipopeptide biosurfactant produced by a marine Bacillus circulans and study its antimicrobial potentials. The marine isolate B. circulans was cultivated in glucose mineral salts medium and the crude biosurfactant was isolated by chemical isolation method. The crude biosurfactants were solvent extracted with methanol and the methanol extract was subjected to reverse phase high-performance liquid chromatography (HPLC). The crude biosurfactants resolved into six major fractions in HPLC. The sixth HPLC fraction eluting at a retention time of 27.3 min showed the maximum surface tension-reducing property and reduced the surface tension of water from 72 mNm(-1) to 28 mNm(-1). Only this fraction was found to posses bioactivity and showed a pronounced antimicrobial action against a panel of Gram-positive and Gram-negative pathogenic and semi-pathogenic micro-organisms including a few multidrug-resistant (MDR) pathogenic clinical isolates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of this antimicrobial fraction of the biosurfactant were determined for these test organisms. The biosurfactant was found to be active against Gram-negative bacteria such as Proteus vulgaris and Alcaligens faecalis at a concentration as low as 10 microg ml(-1). The biosurfactant was also active against methicillin-resistant Staphylococcus aureus (MRSA) and other MDR pathogenic strains. The chemical identity of this bioactive biosurfactant fraction was determined by post chromatographic detection using thin layer chromatography (TLC) and also by Fourier transform infrared (FTIR) spectroscopy. The antimicrobial HPLC fraction resolved as a single spot on TLC and showed positive reaction with ninhydrin, iodine and rhodamine-B reagents, indicating its lipopeptide nature. IR absorption by this fraction also showed similar and overlapping patterns with that of other lipopeptide biosurfactants such as surfactin and lichenysin, proving this biosurfactant fraction to be a lipopeptide. The biosurfactant did not show any haemolytic activity when tested on blood agar plates, unlike the lipopeptide biosurfactant surfactin produced by Bacillus subtilis. The biosurfactant produced by marine B. circulans had a potent antimicrobial activity against Gram-positive and Gram-negative pathogenic and semi-pathogenic microbial strains including MDR strains. Only one of the HPLC fractions of the crude biosurfactants was responsible for its antimicrobial action. The antimicrobial lipopeptide biosurfactant fraction was also found to be nonhaemolytic in nature. This work presents a nonhaemolytic lipopeptide biosurfactant produced by a marine micro-organism possessing a pronounced antimicrobial action against a wide range of bacteria. There is a high demand for new antimicrobial agents because of the increased resistance shown by pathogenic micro-organisms against the existing antimicrobial drugs. This study provides an insight into the search of new bioactive molecules from marine micro-organisms.

Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 microg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6 degrees C, the action time was 7.6 h, and the concentration was 3.46 mg.ml(-1).