Acidic Lipopeptide Research Articles

Spectroscopy Study of Albumin Interaction with Negatively Charged Liposome Membranes: Mutual Structural Effects of the Protein and the Bilayers.

Liposomes as drug carriers are usually injected into the systemic circulation where they are instantly exposed to plasma proteins. Liposome-protein interactions can affect both the stability of liposomes and the conformation of the associated protein leading to the altered biodistribution of the carrier. In this work, mutual effects of albumin and liposomal membrane in the course of the protein's adsorption were examined in terms of quantity of bound protein, its structure, liposome membrane permeability, and changes in physicochemical characteristics of the liposomes. Fluorescence spectroscopy methods and Fourier transform infrared spectroscopy (ATR-FTIR), which provides information about specific groups in lipids involved in interaction with the protein, were used to monitor adsorption of albumin with liposomes based on egg phosphatidylcholine with various additives of negatively charged lipidic components, such as phosphatidylinositol, ganglioside GM1, or the acidic lipopeptide. Less than a dozen of the protein molecules were tightly bound to a liposome independently of bilayer composition, yet they had a detectable impact on the bilayer. Albumin conformational changes during adsorption were partially related to bilayer microhydrophobicity. Ganglioside GM1 showed preferable features for evading undesirable structural changes.

Cadasides, Calcium-Dependent Acidic Lipopeptides from the Soil Metagenome That Are Active against Multidrug-Resistant Bacteria.

The growing threat of antibiotic resistance necessitates the discovery of antibiotics that are active against resistant pathogens. Calcium-dependent antibiotics are a small family of structurally diverse acidic lipopeptides assembled by nonribosomal peptide synthetases (NRPSs) that are known to display various modes of action against antibiotic-resistant pathogens. Here we use NRPS adenylation (AD) domain sequencing to guide the identification, recovery, and cloning of the cde biosynthetic gene cluster from a soil metagenome. Heterologous expression of the cde biosynthetic gene cluster led to the production of cadasides A (1) and B (2), a subfamily of acidic lipopeptides that is distinct from previously characterized calcium-dependent antibiotics in terms of both overall structure and acidic residue rich peptide core. The cadasides inhibit the growth of multidrug-resistant Gram-positive pathogens by disrupting cell wall biosynthesis in the presence of high concentrations of calcium. Interestingly, sequencing of AD domains from diverse soils revealed that sequences predicted to arise from cadaside-like gene clusters are predominantly found in soils containing high levels of calcium carbonate.

Influence of stabilizing components on the integrity of antitumor liposomes loaded with lipophilic prodrug in the bilayer.

Previously, we proposed a liposomal formulation of melphalan (Mlph)-a chemotherapeutic alkylating agent-incorporated in a fluid lipid bilayer in the form of dioleoylglyceride ester. In this work, we compared the stabilizing effect of different amphiphiles included in the Mlph-liposomes, such as phosphatidylinositol (PI), ganglioside GM1, a conjugate of N-carboxymethyl-modified oligoglycine with dioleoylphosphatidylethanolamine (acidic lipopeptide), and polyethylene glycol (2000 Da) conjugated with dipalmitoylphosphatidylethanolamine (PEG-lipid), upon incubation in human serum. Mean hydrodynamic diameter values (86-90 nm) were similar among different liposome samples, while zeta potential values considerably varied. The formulations were incubated in human serum at 37 °C for different time intervals up to 24 h. Liposome integrity was evaluated by changes in fluorescence upon leakage of calcein or disruption of Förster resonance energy transfer between donor and acceptor fluorescent lipid probes in the bilayer. The best stabilization of liposomes was achieved upon the addition of ganglioside GM1 or the acidic lipopeptide. Inclusion of 10 mol% PI improved liposome stability only for the first 4 h of incubation. Pegylated liposomal formulations of melphalan lipophilic prodrug with fluid phase bilayer were the least stable, which is probably due to the propensity of the PEG-lipid to exit liposome membranes. Cholesterol-containing bilayers of liquid ordered phase, supplemented with sufficient amounts of the PEG-lipid, showed good stability in serum.

A liquid chromatography–mass spectrometric method for the detection of cyclic β-amino fatty acid lipopeptides

Bacterial lipopeptides, which contain β-amino fatty acids, are an abundant group of bacterial secondary metabolites exhibiting antifungal and/or cytotoxic properties. Here we have developed an LC-HRMS/MS method for the selective detection of β-amino fatty acid containing cyclic lipopeptides. The method was optimized using the lipopeptides iturin A and puwainaphycin F, which contain fatty acids of similar length but differ in the amino acid composition of the peptide cycle. Fragmentation energies of 10–55eV were used to obtain the amino acid composition of the peptide macrocycle. However, fragmentation energies of 90–130eV were used to obtain an intense fragment specific for the β-amino fatty acid (CnH2n+2N+). The method allowed the number of carbons and consequently the length of the β-amino fatty acid to be estimated. We identified 21 puwainaphycin variants differing in fatty acid chain in the crude extract of cyanobacterium Cylindrospermum alatosporum using this method. Analogously 11 iturin A variants were detected. The retention time of the lipopeptide variants showed a near perfect linear dependence (R2=0.9995) on the length of the fatty acid chain in linear separation gradient which simplified the detection of minor variants. We used the method to screen 240 cyanobacterial strains and identified lipopeptides from 8 strains. The HPLC-HRMS/MS method developed here provides a rapid and easy way to detecting novel variants of cyclic lipopeptides.

A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL) synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin

Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic.

Action of Daptomycin on Membranes

Daptomycin is a clinically important 13 amino acid lipopeptide antibiotic. Its N-terminus is acylated with n-decanol and its C-terminal 10 amino acids form a ring. It is structurally and size-wise different from other well-known membrane-active antimicrobials. Daptomycin is known to disrupt the cytoplasmic membrane function of Gram-positive bacteria by causing leakage of potassium (and potentially other) ions, leading to the loss of membrane potential and cell death. The critical factor affecting the function of daptomycin is its interaction with negatively charged lipids such as PG in a calcium (Ca++) dependent manner. Based on previous research on cell membranes, daptomycin has been assumed to insert and aggregate in the membrane, and then to alter the membrane curvature. However these details have not been demonstrated by biophysical studies. In our aspirated GUV experiments, we found that with a DOPG-containing GUV and a sufficient concentration of Ca++, daptomycin can extract lipid molecules, and form lipid-peptide aggregations. The lipid-peptide aggregates did not occur if cardiolipin replaced PG, or if other divalent ions, such as Mg++, Ba++ replaced Ca++. Similarly, daptomycin did not bind to a GUV when cardiolipin substituted for DOPG or in the absence of Ca++. Daptomycin with Ca++ did bind to a pure DOPC GUV, but had no other effects. Furthermore, with the presence of daptomycin, DOPG and Ca++, we found Ca++ permeates into the GUV, while a content dye, Texas red dextran, did not leak out. This result suggests that daptomycin and Ca++ do not form pores on the membrane of DOPG-contained GUV, but cause leakage of ions. Finally, daptomycin with DOPG and Ca++ produces a negative exciton CD couplet centered at the 225 nm absorption peak of Trptophan1 and Kynurenine13, whereas in all other conditions, the exciton CD couplet is positive.

Diaminopropionic acid lipopeptides: Characterization studies of polyplexes aimed at pDNA delivery

Here we report a novel class of peptides—d-diaminopropionic acids (Dap)—for gene delivery. These peptides have attractive properties for gene delivery, and the advantage that they can be easily manipulated in relation to their composition, abiding with tailored-design. We characterized the toxicological and biophysical properties of DNA particles resulting from the interaction of the nucleic acid with a series of Dap8 peptides conjugated to different alkyl groups. These peptides formed small and homogenous DNA particle populations that protected against DNase I degradation at non-toxic concentrations. However, despite the similarity between these peptides and others that are arginine-rich, and efficient vectors, functional studies suggest the need for additional modifications in the carriers to improve their DNA delivery efficiency. Taken together, these studies underscore the relevance of the overall structure of the carrier and the complexity of designing from scratch a carrier.

Exploring the mechanism of lipid transfer during biosynthesis of the acidic lipopeptide antibiotic CDA

The non-ribosomally synthesized lipodepsipeptide CDA belongs to the group of acidic lipopeptide antibiotics, whose members feature a fatty acid side chain that strongly affects their antimicrobial activity. This study elucidates the N-acylation of the N-terminal serine in the CDA peptide chain. This reaction is referred to as lipoinitiation and is shown to be catalyzed by the dissected starter C domain found at the N-terminus of Cda-PSI. The recombinantly produced C domain specifically interacts with 2,3-epoxyhexanoyl-S-ACP and catalyzes the transfer of the fatty acid moiety onto the amino group of PCP-bound serine with high selectivity for both carrier protein bound substrates at the donor and acceptor site.

Biosynthesis and biosynthetic engineering of calcium-dependent lipopeptide antibiotics

Abstract Biosynthetic engineering involves the reprogramming of genes that are involved in the biosynthesis of natural products to generate new "non-natural" products, which might otherwise not exist in nature. Potentially this approach can be used to provide large numbers of secondary metabolites variants, with altered biological activities, many of which are too complex for effective total synthesis. Recently we have been investigating the biosynthesis of the calcium-dependent antibiotics (CDAs) which are members of the therapeutically relevant class of acidic lipopeptide antibiotics. CDAs are assembled by nonribosomal peptide synthetase (NRPS) enzymes. These large modular assembly-line enzymes process intermediates that are covalently tethered to peptidyl carrier protein (PCP) domain bonds bonds, which makes them particularly amenable to reprogramming. The CDA producer, Streptomyces coelicolor, is also a genetically tractable model organism which makes CDA an ideal template for biosynthetic engineering. To this end we have elucidated many of the key steps in CDA biosynthesis and utilized this information to develop methods that have enabled the engineered biosynthesis of wide range of CDA-type lipopeptides.

The Structural Diversity of Acidic Lipopeptide Antibiotics

Acidic lipopeptide antibiotics are a new class of potent antibiotics, which includes daptomycin, A54145, calcium-dependent antibiotics (CDAs), friulimicins/amphomycins, laspartomycin/glycinocins and others. The importance of this novel class is exemplified by the success story of the clinically approved daptomycin, which is used for the treatment of skin infections and bacteremia caused by multidrug-resistant bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The potency of acidic lipopeptides is inherent in their chemical structure. The nonribosomally synthesized peptide cores consist of eleven to 13 amino acids, which are rigidified by the formation of a ten-membered ring. An N-terminal fatty acid, which facilitates insertion into the lipid bilayer of bacterial membranes, completes the structure. All these antibiotics contain multiple nonproteinogenic amino acids as well as different lipid tails; this yields remarkable structural diversity. This review summarizes the observed structural variety through a detailed description of the composition of the acidic lipopeptides. Furthermore, engineering approaches towards novel lipopeptides are presented. Recent discoveries in the field of tailoring enzymes, which enable structural plurality mainly by amino and fatty acid precursor biosynthesis, are highlighted.

Antibacterial Activity of Ultrashort Cationic Lipo-β-Peptides

Previously reported D,L-lipo-alpha-peptides and their lipo-beta-peptide counterparts (C16-KGGK, C16-KAAK, C16-KKKK, and C12-KLLK) were studied, and the lipo-beta-peptides were found to retain antimicrobial activity. Likewise, no significant changes in antimicrobial activity were found upon activity comparisons with D,L-amino acid-based lipopeptides or any L-amino acid lipopeptides. As a defined amphipathic structure is unlikely to form with such short molecules and as similar activities were obtained from all lipopeptides, we suspect that the action of membrane permeation is retained.

Surfactin-Triggered Small Vesicle Formation of Negatively Charged Membranes: A Novel Membrane-Lysis Mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri=200) and is complete for Ri=50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Stereospecific Enzymatic Transformation of α-Ketoglutarate to (2S,3R)-3-Methyl Glutamate during Acidic Lipopeptide Biosynthesis

The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues DptI from the daptomycin producer Streptomyces roseosporus and LptI from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, DptI, and LptI are S-adenosyl methionine (SAM)-dependent alpha-ketoglutarate methyltransferases that catalyze the stereospecific methylation of alpha-ketoglutarate (alphaKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IlvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of alphaKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of beta-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.

Mechanistic and Structural Basis of Stereospecific Cβ-Hydroxylation in Calcium-Dependent Antibiotic, a Daptomycin-Type Lipopeptide

Non-ribosomally synthesized lipopeptide antibiotics of the daptomycin type are known to contain unnatural beta-modified amino acids, which are essential for bioactivity. Here we present the biochemical and structural basis for the incorporation of 3-hydroxyasparagine at position 9 in the 11-residue acidic lipopeptide lactone calcium-dependent antibiotic (CDA). Direct hydroxylation of l-asparagine by AsnO, a non-heme Fe(2+)/alpha-ketoglutarate-dependent oxygenase encoded by the CDA biosynthesis gene cluster, was validated by Fmoc derivatization of the reaction product and LC/MS analysis. The 1.45, 1.92, and 1.66 A crystal structures of AsnO as apoprotein, Fe(2+) complex, and product complex, respectively, with (2S,3S)-3-hydroxyasparagine and succinate revealed the stereoselectivity and substrate specificity of AsnO. The comparison of native and product-complex structures of AsnO showed a lid-like region (residues F208-E223) that seals the active site upon substrate binding and shields it from sterically demanding peptide substrates. Accordingly, beta-hydroxylated asparagine is synthesized prior to its incorporation into the growing CDA peptide. The AsnO structure could serve as a template for engineering novel enzymes for the synthesis of beta-hydroxylated amino acids.

Chemoenzymatic Design of Acidic Lipopeptide Hybrids: New Insights into the Structure−Activity Relationship of Daptomycin and A54145

The acidic lipopeptides, including the clinically approved antibiotic daptomycin, constitute a class of structurally related branched cyclic peptidolactones and peptidolactams synthesized by nonribosomal peptide synthetases (NRPSs). In this study, the excised peptide cyclases from A54145 and daptomycin NRPSs were shown to be able to catalyze the macrocyclization of peptide thioester substrates, which were chemically produced by solid phase peptide synthesis. Applying this chemoenzymatic strategy, we generated derivatives of A54145 and daptomycin as well as hybrid molecules of both compounds. Bioactivity determination of the derived cyclic molecules revealed new insights into the structure-activity relationship of the acidic lipopeptide family. The general importance of several amino acid positions, including two conserved aspartic acid residues, was confirmed to be substantial for antibiotic potency. As a robust macrocyclization catalyst, the peptide cyclase excised from A54145 synthetase is the first cyclase of a branched cyclic lipopeptide, which catalyzes both macrolactonization and macrolactamization. The results presented herein illustrate the advantages of combining organic synthesis with natural product biosynthetic enzymes to explore the interplay between structural features and biological activity.

An electrochemical study on the interaction of surfactin with a supported bilayer lipid membrane on a glassy carbon electrode

Surfactin is an acidic lipopeptide with amphiphilic character, which can interact with a biomembrane. The interaction of surfactin with a supported bilayer lipid membrane on a glassy carbon electrode (GCE) was investigated by cyclic voltammetry and ac impedance spectroscopy in this paper. Surfactin could induce pores in the bilayer lipid membrane and even cause the destruction of the membrane. The mechanism of the interaction of surfactin with the supported bilayer lipid membrane was studied. The insertion of surfactin into the lipid membrane was the first step in the formation of pores. Subsequently, surfactin aggregated in the lipid membrane to form the pores. Along with the transition of surfactin molecules from the outer to the inner layer, which was connected to the surface of the GCE, the lipid membrane was destroyed.

The anti-thrombotic activity of surfactins

Platelet aggregation was inhibited and the density of platelet-rich plasma (PRP) clots was decreased by the preincubation of PRP with surfactins, an acidic lipopeptide of Bacillus subtilis complex BC1212 isolated from soybean paste, in dose-dependent manner. Our findings suggest that surfactins are able to prevent a platelet aggregation leading to an inhibition of additional fibrin clot formation, and to enhance fibrinolysis with facilitated diffusion of fibrinolytic agents.

Synthesis and Derivatization of Daptomycin: A Chemoenzymatic Route to Acidic Lipopeptide Antibiotics

Daptomycin is a branched cyclic nonribosomally assembled acidic lipopeptide, which is the first clinically approved antibiotic of this class. Here we show that the recombinant cyclization domain of the Streptomyces coelicolor calcium-dependent antibiotic (CDA) nonribosomal peptide synthetase (NRPS) is a versatile tool for the chemoenzymatic generation of daptomycin derivatives. Linear CDA undecapeptide thioesters with single exchanges at six daptomycin-specific residues were successfully cyclized by CDA cyclase. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues yielded a daptomycin derivative that lacked only the beta-methyl group of l-3-methylglutamate. Bioactivity studies with several substrate analogues revealed a significant role of nonproteinogenic constituents for antibacterial potency. In accordance with acidic lipopeptides, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the concentration of calcium ions. Single deletions of the four acidic residues in the peptide backbone suggest that only two aspartic acid residues are essential for antimicrobial potency. These two residues are strictly conserved among other nonribosomal acidic lipopeptides and the EF-motif of ribosomally assembled calmodulin. Based on these findings CDA cyclase is a versatile catalyst that can be used to generate novel daptomycin derivatives that are otherwise difficult to obtain by chemical modification of the parental tridecapeptide to improve further its therapeutic activity.

Chemo- and regioselective peptide cyclization triggered by the N-terminal fatty acid chain length: the recombinant cyclase of the calcium-dependent antibiotic from Streptomyces coelicolor.

Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr(2) and Ser(1) residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser(1) or Thr(2) by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only l-configured Ser(1) and Thr(2) for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C(2) to C(6) as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.