Screening concepts,characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

Lipopeptide biosurfactants are surface active biomolecules that are produced by a variety of microorganisms. Microbial lipopeptides have gained the interest of microbiologists, chemists and biochemists for their high biodiversity as well as efficient action, low toxicity and good biodegradability in comparison to synthetic counterparts. In this report, we review methods for the production, isolation and screening, purification and structural characterization of microbial lipopeptides. Several techniques are currently available for each step, and we describe the most commonly utilized and recently developed techniques in this review. Investigations on lipopeptide biosurfactants in natural products require efficient isolation techniques for the characterization and evaluation of chemical and biological properties. A combination of chromatographic and spectroscopic techniques offer opportunities for a better characterization of lipopeptide structures, which in turn can lead to the application of lipopeptides in food, pharmaceutical, cosmetics, agricultural and bioremediation industries.     

芽孢杆菌几种重要抗菌脂肽研究进展

多种芽孢杆菌为益生菌,能分泌多种天然抗菌活性物质,其中脂肽是重要的一类。目前已鉴定的脂肽约有90多种,多数为环脂肽。脂肽中表面活性素(surfactin)、伊枯草菌素(iturin)、芬原素(fengycin)、杆菌霉素(bacillomycin)、多粘菌素(polymyxins)等是研究最广泛的脂肽。其中surfactin、iturin、fengycin由于其具有表面活性剂特性及抗真菌、抗细菌、抗病毒、抗肿瘤、抗炎症等功能,应用潜力巨大。本文对surfactin、iturin及fengycin的结构、功能、合成调控及其分离纯化和生产等方面的研究进展进行了评述。合成生物学是提高脂肽产量的重要手段,未来脂肽可用于种植业、养殖业、食品、医药、石油工业和环保等领域,因此需要在新型脂肽的发现、高产活性脂肽的生产、脂肽低廉生产技术的研发及安全性的评估等方面加强研究。     

Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm formation of Candida albicans

Rhamnolipids are biodegradable low toxic biosurfactants which exert antimicrobial and anti-biofilm properties. They have attracted much attention recently due to potential applications in areas of bioremediation, therapeutics, cosmetics and agriculture, however, the full potential of these versatile molecules is yet to be explored. Based on the facts that many naturally occurring lipopeptides are potent antimicrobials, our study aimed to explore the potential of replacing rhamnose in rhamnolipids with amino acids thus creating lipopeptides that would mimic or enhance properties of the parent molecule. This would allow not only for more economical and greener production but also, due to the availability of structurally different amino acids, facile manipulation of physico-chemical and biological properties.Our synthetic efforts produced a library of 43 lipopeptides revealing biologically more potent molecules. The structural changes significantly increased, in particular, anti-biofilm properties against Candida albicans, although surface activity of the parent molecule was almost completely abolished. Our findings show that the most active compounds are leucine derivatives of 3-hydroxy acids containing benzylic ester functionality. The SAR study demonstrated a further increase in activity with aliphatic chain elongation. The most promising lipopeptides 15, 23 and 36 at 12.5 µg/mL concentration allowed only 14.3%, 5.1% and 11.2% of biofilm formation, respectively after 24 h. These compounds inhibit biofilm formation by preventing adhesion of C. albicans to abiotic and biotic surfaces.     

Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI‐TOF‐MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1‐diphenyl‐2‐picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration‐dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of β‐carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram‐positive, Gram‐negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional‐food components.     

Production of mannosylerythritol lipids and their application in cosmetics

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants abundantly produced by different basidiomycetous yeasts such as Pseudozyma, and show not only excellent interfacial properties but also versatile biochemical actions. These features of MELs make their application in new technology areas possible. Recently, the structural and functional variety of MELs was considerably expanded by advanced microbial screening methods. Different types of MELs bearing different hydrophilic and hydrophobic parts have been reported. The genes responsible for MEL biosynthesis were identified, and their genetic study is now in progress, aiming to control the chemical structure. The excellent properties leading to practical cosmetic ingredients, i.e., moisturization of dry skin, repair of damaged hair, activation of fibroblast and papilla cells and antioxidant and protective effects in skin cells, have been demonstrated on the yeast glycolipid biosurfactants. In this review, the current status of research and development on MELs, particularly the commercial application in cosmetics, is described.     

Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis

The structural analysis of post‐translational modifications (PTMs) of lipoproteins is difficult due to the hydrophobic properties of their fatty acid moieties. At the present time, the relative positions of fatty acid components on the N‐acyl‐S‐diacylglycerylcysteine core structure has not been specifically identified in any natural or bacterial expressed recombinant lipoproteins. In this study, we describe a rapid solid‐phase extraction using acetonitrile and isopropanol method that can be performed manually to isolate large amounts of relatively pure lipopeptides generated by the limited tryptic‐digestion of recombinant lipoproteins. Using these lipopeptides and LC/MS mass spectra analysis, two groups of N‐terminal lipidated (diacyl or triacyl) molecules that differ by one fatty acid unit were successfully identified. This LC/MS method also provided the separation of lipopeptides differing by 14 Da for the on‐line MS identification. Multiple‐stage fragmentation analyses of the di‐ and triacyl lipopeptides using both the positive and negative ion modes enabled to identify the putative structure of the N‐acyl‐S‐diacylglycerylcysteine containing an amide bond to palmitic acid at the N‐terminal cysteine, a palmitic acid at sn1 position, and an unsaturated fatty acid of either hexadecenoic acid, cyclopropaneoctanoic acid, oleic acid and nonadecenoic acid at sn2 position of diacylglycerol residue through ester bonding. For diacyl lipoprotein, the saturated palmitoyl fatty acid group is absent at sn1 position of glycerol‐derived lipid residue of lipopeptide.     

Synthesis of thiol-reactive lipopeptide adjuvants. Incorporation into liposomes and study of their mitogenic effect on mouse splenocytes

Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of importance for the further optimization of antigen-specific liposomal-based synthetic vaccines; the bromoacetyl derivative of Pam(3)CAG should be a promising lipopeptide derivative serving as an anchor for peptide epitopes while retaining its lymphocyte activation activity.     

Are the short cationic lipopeptides bacterial membrane disruptors? Structure-Activity Relationship and molecular dynamic evaluation

Short cationic lipopeptides are amphiphilic molecules that exhibit antimicrobial activity mainly against Gram-positives. These compounds bind to bacterial membranes and disrupt their integrity. Here we examine the structure-activity relation (SAR) of lysine-based lipopeptides, with a prospect to rationally design more active compounds. The presented study aims to explain how antimicrobial activity of lipopeptides is affected by the charge of lipopeptide headgroup and the length of lipopeptide acyl chain. The obtained SAR models suggest that the lipophilicity of short synthetic cationic lipopeptides is the major factor that determines their antimicrobial activities. In order to link the differences in antimicrobial activity to the mechanism of action of lipopeptides containing one and two hydrophobic chains, we additionally performed molecular dynamic (MD) simulations. By using combined coarse-grained and all-atom simulations we also show that these compounds neither affect the organization of the membrane lipids nor aggregate to form separate phases. These results, along with the onset of antimicrobial activity of lipopeptides well below the critical micelle concentration (CMC), indicate that lipopeptides do not act in a simple detergent-like manner.     

Physicochemical and biological analysis of synthetic bacterial lipopeptides: validity of the concept of endotoxic conformation

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle.     

An update on the use of unconventional substrates for biosurfactant production and their new applications

Biosurfactants are valuable microbial amphiphilic molecules with effective surface-active and biological properties applicable to several industries and processes. Microbes synthesize them, especially during growth on water-immiscible substrates, providing an alternative to chemically prepared conventional surfactants. Because of their structural diversity (i.e., glycolipids, lipopeptides, fatty acids, etc.), low toxicity, and biodegradability, these molecules could be widely used in cosmetic, pharmaceutical, and food processes as emulsifiers, humectants, preservatives, and detergents. Moreover, they are ecologically safe and can be applied in bioremediation and waste treatments. They can be produced from various substrates, mainly renewable resources such as vegetable oils, distillery and dairy wastes, which are economical but have not been reported in detail. In this review, we report advances made in using renewable substrates for biosurfactant production and their newer applications.     

芽胞杆菌产生的脂肽类抗生素的结构和应用

脂肽类抗生素不仅对细菌、真菌有抗菌作用,而且对支原体、寄生虫和病毒等也具有显著的抑制作用。其主要包括表面活性素(Surfactins)、伊枯草菌素(Iturins)和芬芥素(Fengycins),其中Iturins和Fengycins有抗真菌活性,Surfactins有抗支原体、抗病毒等活性。综述了芽胞杆菌产生的脂肽类抗生素的结构、功能和应用,为其在农业和医药的研究与应用提供参考。     

合成生物学指导下芽孢杆菌合成环脂肽的研究进展*

近年来,合成生物学借助工程化在人工生命系统的设计与构建方面取得了长足进展,特别是“细胞工厂”的开发和应用为天然产物的合成带来了深刻变革。环脂肽是一类新型的天然表面活性剂,因其特殊的结构和功能亦可作为抗生素使用。目前,合成环脂肽最理想的微生物底盘是芽孢杆菌。因此,许多研究者致力于通过合成生物学技术来提升芽孢杆菌作为环脂肽细胞工厂的性能。首先,对芽孢杆菌中环脂肽的非核糖体肽合成途径进行概述;其次,重点介绍与环脂肽合成相关的调控因子;再次,从底盘细胞的选择、基因编辑工具的开发、合成路径的优化及发酵过程的优化等四个方面对合成生物学指导下环脂肽的相关研究进展进行总结;最后,讨论环脂肽合成中可能存在的挑战,并就未来研究趋势进行展望,以期为高效环脂肽细胞工厂的开发提供参考。     

Biological activity of lipopeptides from <Emphasis Type="Italic">Bacillus</Emphasis>

The lipopeptides of Bacillus are small metabolites that contain a cyclic structure formed by 7–10 amino acids (including 2–4 d-amino acids) and a beta-hydroxy fatty acid with 13–19 C atoms. These lipopeptides exhibit a variety of biological activities, including interactions with biofilms, and anti-fungal, anti-inflammatory, anti-tumor, anti-virus, and anti-platelet properties. The multiple activities of lipopeptides have stimulated significant interest in the exploitation of these lipopeptides for use as antibiotics, feed additives, anti-tumor agents, urgent thrombolytic therapeutic agents, and drug delivery systems. Understanding the natural function of these structurally diverse lipopeptides in Bacillus provides insight into microbial regulatory programs and is required for efficient development of more effective products. Currently, there is still insufficient knowledge of the direct target of these lipopeptides, and continued efforts are needed to enhance their biosynthesis efficiency for industrial applications.     

Design and study of lipopeptide inhibitors on preventing aggregation of human islet amyloid polypeptide residues 11‐20

Type 2 diabetes mellitus, a kind of conformational disease, has become an epidemic disease, which seriously endangers the quality of life and health of human beings. The deposition of human islet amyloid polypeptide (hIAPP) has been considered as one of the major pathological features of type 2 diabetes mellitus. As lipopeptides have some hydrophobic groups, which are similar to the reported aggregation inhibitors, and some lipopeptides could prevent cells from depositing of amyloid fibrils, several potential lipopeptide inhibitors have been engineered and synthesized, which have been assessed for their inhibitory effect in preventing amyloid fibrils formation of hIAPP_11‐20 by using the conventional thioflavin‐T fluorescence assay and new technique microscale thermophoresis (MST). The final amyloid fibrils of hIAPP_11‐20 were characterized by transmission electron microscopy. Results suggested that with the increasing length of alkyl chain, the antiaggregation efficiency of lipopeptide inhibitors towards hIAPP_11‐20 increased gradually. Meanwhile, the amount of arginines, which represent the head groups of lipopeptides, may also have some influence. The binding events also showed that the inhibitory efficiency of these lipopeptide inhibitors was enhanced with the increase of affinities between lipopeptides and hIAPP_11‐20, which were obtained from MST. This study demonstrated the efficiency of lipopeptides in inhibiting the aggregation of hIAPP_11‐20 and proved that MST could be regarded as an appropriate and rapid method to screen potential inhibitors of hIAPP_11‐20 or other amyloid proteins. This study also broadens the types of inhibitors on inhibiting amyloid formation of hIAPP.     

Biological and structural effects of the conjugation of an antimicrobial decapeptide with saturated,unsaturated, methoxylated and branched fatty acids

The increasing bacterial resistance against conventional antibiotics has led to the search for new antimicrobial drugs with different modes of action. Cationic antimicrobial peptides (AMPs) and lipopeptides are promising candidates to treat infections because they act on bacterial membranes causing rapid destruction of sensitive bacteria. In this study, a decapeptide named A2 (IKQVKKLFKK) was conjugated at the N‐terminus with saturated, unsaturated, methoxylated and methyl ‐branched fatty acids of different chain lengths (C8 – C20), the antimicrobial and structural properties of the lipopeptides being then investigated. The attachment of the fatty acid chain significantly improved the antimicrobial activity of A2 against bacteria, and so, endowed it with moderated antifungal activity against yeast strains belonging to genus Candida. Lipopeptides containing hydrocarbon chain lengths between C8 and C14 were the best antibacterial compounds (MIC = 0.7 to 5.8 μM), while the most active compounds against yeast were A2 conjugated with methoxylated and enoic fatty acids (11.1 to 83.3 μM). The improvement in antimicrobial activity was mainly related to the amphipathic secondary structure adopted by A2 lipopeptides in the presence of vesicles that mimic bacterial membranes. Peptide conjugation with long hydrocarbon chains (C12 or more), regardless of their structure, significantly increased toxicity towards eukaryotic cells, resulting in a loss of selectivity. These findings suggest that A2‐derived lipopeptides are potential good candidates for the treatment of infectious diseases caused by bacteria and opportunistic pathogenic yeast belonging to genus Candida. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of self-assembly on antimicrobial activity of double-chain short cationic lipopeptides

Short cationic antimicrobial lipopeptides with surfactant-like structure are promising antibiotic candidates that preferentially target microbial membranes. Therefore, we focused our study on double-chain lipopeptides, (C_10-16)₂Dab-KKK-NH₂ and (C_10-16)₂Dap-KKK-NH₂, where Dab and Dap are 2,4-diaminobutyric and 2,3-diaminopropionic acids, respectively. We tried to answer a question how the self-assembly behaviour affects biological activities of the tested compounds. The subject compounds were synthesized by solid-phase method and screened for their antimicrobial and haemolytic activities. Cytotoxicity tests on human keratinocytes were carried out for the most promising lipopeptides. Self-assembly properties were evaluated by both experimental and theoretical methods. Interactions with membrane models were examined using the ITC and FTIR techniques. All the lipopeptides studied showed the tendency to self-assembly in solution, and this behaviour was affected by the length of the hydrocarbon chains. Acyl chain elongation supported the formation of the bilayer structure and deprived the lipopeptides of antimicrobial activity. A multi-step mechanism of interaction with a negatively charged membrane was observed for the short-chain lipopeptides, indicating other processes accompanying the binding process. Short-chain lipopeptides were able to penetrate into the liposome’s interior and/or cause the rupture of the liposome, this being compatible with their high antimicrobial activity.     

Genome mining reveals high incidence of putative lipopeptide biosynthesis NRPS/PKS clusters containing fatty acyl‐AMP ligase genes in biofilm‐forming cyanobacteria

Cyanobacterial lipopeptides have antimicrobial and antifungal bioactivities with potential for use in pharmaceutical research. However, due to their hemolytic activity and cytotoxic effects on human cells, they may pose a health issue if produced in substantial amounts in the environment. In bacteria, lipopeptides can be synthesized via several well‐evidenced mechanisms. In one of them, fatty acyl‐AMP ligase (FAAL) initiates biosynthesis by activation of a fatty acyl residue. We have performed a bioinformatic survey of the cyanobacterial genomic information available in the public databases for the presence of FAAL‐containing non‐ribosomal peptide synthetase/polyketide synthetase (NRPS/PKS) biosynthetic clusters, as a genetic basis for lipopeptide biosynthesis. We have identified 79 FAAL genes associated with various NRPS/PKS clusters in 16% of 376 cyanobacterial genomic assemblies available, suggesting that FAAL is frequently incorporated in NRPS/PKS biosynthetases. FAAL was present either as a stand‐alone protein or fused either to NRPS or PKS. Such clusters were more frequent in derived phylogenetic lineages with larger genome sizes, which is consistent with the general pattern of NRPS/PKS pathways distribution. The putative lipopeptide clusters were more frequently found in genomes of cyanobacteria that live attached to surfaces and are capable of forming microbial biofilms. While lipopeptides are known in other bacterial groups to play a role in biofilm formation, motility, and colony expansion, their functions in cyanobacterial biofilms need to be tested experimentally. According to our data, benthic and terrestrial cyanobacteria should be the focus of a search for novel candidates for lipopeptide drug synthesis and the monitoring of toxic lipopeptide production.     

TLR1- and TLR6-independent recognition of bacterial lipopeptides

Bacterial cell walls contain lipoproteins/peptides, which are strong modulators of the innate immune system. Triacylated lipopeptides are assumed to be recognized by TLR2/TLR1-, whereas diacylated lipopeptides use TLR2/TLR6 heteromers for signaling. Following our initial discovery of TLR6-independent diacylated lipopeptides, we could now characterize di- and triacylated lipopeptides (e.g. Pam(2)C-SK(4), Pam(3)C-GNNDESNISFKEK), which have stimulatory activity in TLR1- and in TLR6-deficient mice. Furthermore, for the first time, we present triacylated lipopeptides with short length ester-bound fatty acids (like PamOct(2)C-SSNASK(4)), which induce no response in TLR1-deficient cells. No differences in the phosphorylation of MAP kinases by lipopeptide analogs having different TLR2-coreceptor usage were observed. Blocking experiments indicated that different TLR2 heteromers recognize their specific lipopeptide ligands independently from each other. In summary, a triacylation pattern is necessary but not sufficient to render a lipopeptide TLR1-dependent, and a diacylation pattern is necessary but not sufficient to render a lipopeptide TLR6-dependent. Contrary to the current model, distinct lipopeptides are recognized by TLR2 in a TLR1- and TLR6-independent manner.     

Short cationic lipopeptides as effective antibacterial agents: Design,physicochemical properties and biological evaluation

The spread of drug-resistant bacteria has imparted a sense of urgency in the search for new antibiotics. In an effort to develop a new generation of antibacterial agents, we have designed de novo charged lipopeptides inspired by natural antimicrobial peptides. These short lipopeptides are composed of cationic lysine and hydrophobic lipoamino acids that replicate the amphiphilic properties of natural antimicrobial peptides. The resultant lipopeptides were found to self-assemble into nanoparticles. Some were effective against a variety of Gram-positive bacteria, including strains resistant to methicillin, daptomycin and/or vancomycin. The lipopeptides were not toxic to human kidney and liver cell lines and were highly resistant to tryptic degradation. Transmission electron microscopy analysis of bacteria cells treated with lipopeptide showed membrane-damage and lysis with extrusion of cytosolic contents. With such properties in mind, these lipopeptides have the potential to be developed as new antibacterial agents against drug-resistant Gram-positive bacteria.     

Lipopeptides in microbial infection control: Scope and reality for industry

Lipopeptides are compounds that are formed by cyclic or short linear peptides linked with a lipid tail or other lipophilic molecules. Recently, several lipopeptides were characterized, showing surfactant, antimicrobial and cytotoxic activities. The properties of lipopeptides may lead to applications in diverse industrial fields including the pharmaceutical industry as conventional antibiotics; the cosmetic industry for dermatological product development due to surfactant and anti-wrinkle properties; in food production acting as emulsifiers in various foodstuffs; and also in the field of biotechnology as biosurfactants. Some lipopeptides have reached a commercial antibiotic status, such as daptomycin, caspofungin, micafungin, and anidulafungin. This will be the focus of this review. Moreover, the review presented here will focus on the biotechnological utilization of lipopeptides in different fields as well as the functional–structure relation, connecting recent aspects of synthesis and structure diversity.