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.     

A novel lipopeptide produced by a Pacific Ocean deep-sea bacterium, Rhodococcus sp. TW53

Aims: Our goal was to find a novel, biosurfactant‐producing bacterium from Pacific Ocean deep‐sea sediments. Methods and Results: An oil‐degrading biosurfactant‐producing bacterium TW53 was obtained from deep‐sea sediment, and was identified through 16S rDNA analysis as belonging to the genus Rhodococcus. It lowered the surface tension of its culture to 34·4 mN m^?1. Thin layer chromatography (TLC) showed that the crude biosurfactants of TW53 were composed of lipopeptides and free fatty acids (FA). The lipopeptides were purified with column chromatography and then hydrolysed with 6 mol l^?1 HCl. Gas chromatography‐mass spectrometry analysis showed that the hydrolyte in the hydrophobic fraction contained five kinds of FA with chain lengths of C₁₄–C₁₉, and C₁₆H₃₂O₂ was a major component making up 59·18% of the total. However, 3‐hydroxyl FA was not found, although it is usually found in lipopeptides. Silica gel TLC revealed that the hydrolyte in the hydrophilic fraction was composed of five kinds of amino acids; consistently, ESI‐Q‐TOF‐MS analysis confirmed the composition results and provided their sequence tentatively as Ala‐Ile‐Asp‐Met‐Pro. Furthermore, the yield and CMC (critical micelle concentrations) of purified lipopeptides were examined. The purified product reduced the surface tension of water to 30·7 mN m^?1 with a CMC value of 23·7 mg l^?1. These results suggest that Rhodococcus sp. TW53 produces a novel lipopeptide that we have named rhodofactin. Conclusion: The deep‐sea isolate Rhodococcus sp. TW53 was the first reported lipopeptide‐producing bacterium of this genus. The lipopeptides had novel chemical compositions. Significance and Impact of the Study: Rhodococcus sp. TW53 has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution.     

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.     

Increase of anti-HIV activity of C-peptide fusion inhibitors using a bivalent drug design approach

We reported the design of fusion inhibitors with improved activity using a multivalent inhibitor design strategy. First, we chose C29 as the template sequence, which is a 29-mer peptide derived from HIV-1 gp41 CHR domain and has anti-HIV activity of IC₅₀ 118 nM in a cell–cell fusion assay. We optimized the crosslink sites and linkers of the template peptide. We found that N-terminal crosslink caused activity improvement based on the multivalent co-operative effect. Especially, the IC₅₀ of peptide (CAcaC29)₂ was improved from 49.02 (monomeric form) to 5.71 nM. Compared with long peptides, short peptides may be more suitable to analyze the co-operative effect. So we selected a shorter peptide C22 to synthesize the bivalent inhibitors. Due its weak helicity, no co-operative effect appeared. Therefore, we chose SC22EK, which were introduced salt bridges to consolidate the helicity based on the natural sequence C22. The cross-linked (CAcaSC22EK)₂ was four times more potent than the monomer SC22EK in anti-HIV activity, with an IC₅₀ value of 4.92 nM close to the high active peptide fusion inhibitor C34. The strategy used in this study may be used to design new fusion inhibitors to interfere similar processes.     

Structure–activity relationships in ultrashort cationic lipopeptides: the effects of amino acid ring constraint on antibacterial activity

Taking a minimalistic approach in efforts to lower the cost for the development of new synthetic antimicrobial peptides, ultrashort cationic lipopeptides were designed to mimic the amphiphilic nature crucial for their activity but with only a very short peptide sequence ligated to a lipidic acid. Nine ultrashort cationic lipopeptides were prepared to study the effects of ring constraint in the amino acid side chain of the peptide component. USCL-P_Cat1, consisting of only four l-4R-aminoproline residues and acylated with palmitic acid at the N-terminus, was found to populate a polyproline II helical secondary conformation that is stable to different pHs and temperatures using circular dichroism. The synthesized lipopeptides were found to have a micellar structure in water using negative staining transmission electron microscopy. We found that constraining the side chain of the amino acid component is not beneficial to the antimicrobial activity. USCL-Dab1, USCL-Dab3 and USCL-K1 showed promising activity against a panel of laboratory reference and clinically isolated Gram-positive and Gram-negative bacterial strains, some of which are multidrug resistant. No appreciable cytotoxicity against human monocytic THP-1 cells was observed up to concentrations of 20–40 µM for all synthesized compounds. Moreover, all USCLs did not induce the production of either pro-inflammatory cytokines or chemokines up to 40 µM.     

Selective fengycin production in a modified rotating discs bioreactor

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient k _L a were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787 mg L^?1), but also better selectivity of the bioreaction towards fengycin (up to 88 %) and better yields of fengycin per glucose (up to 62.9 mg g^?1), lipopeptides per glucose (up to 71.5 mg g^?1), fengycin per biomass (up to 309 mg g^?1) and lipopeptides per biomass (up to 396 mg g^?1) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30 min^?1. The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.     

Short arginine-rich lipopeptides: From self-assembly to antimicrobial activity

In this paper, we examine antimicrobial and cytotoxic activities, self-assembly and interactions with anionic and zwitterionic membranes of short arginine-rich lipopeptides: C₁₆-RRRR-NH₂, C₁₄-RRRR-NH₂, C₁₂-RRRR-NH₂, and C₁₆-PRRR-NH₂. They show a tendency to self-assembly into micelles, but it is not required for antimicrobial activity. The membrane binding of the lipopeptides can be accompanied by other factors such as: peptide aggregation, pore formation or micellization of phospholipid bilayer. The shortening of the acyl chain results in compounds with a lower haemolytic activity and a slightly improved antimicrobial activity against Gram-positive bacteria, what indicates enhanced cell specificity. Results of coarse-grained molecular dynamics simulations indicate different organization of membrane lipids upon binding of arginine-based lipopeptides and the previously studied lysine-based ones.     

Human Integrin α3β1 Regulates TLR2 Recognition of Lipopeptides from Endosomal Compartments

Background

Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered.

Methodology/Principal Findings

Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam₃CSK₄, are dependent upon an integrin, α₃β₁. The mechanism for integrin α₃β₁ involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam₃CSK₄ is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane.

Conclusion/Significance

Here we identify integrin α₃β₁ as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α₃β₁-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α₃β₁ serves as a mechanism for modulating inflammatory responses.     

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。     

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.     

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.     

假单胞菌合成环脂肽转录调控的研究进展

假单胞菌所合成的环脂肽是一类由环状的寡肽连接一个脂肪酸链组成的两亲性分子,利用巯基化模块由非核糖体肽合成酶合成。环脂肽的生物合成受到严格、复杂的调控,GacS/GacA双组分系统和群体感应系统是其中两类重要的调控系统。本文总结假单胞菌合成环脂肽的调控机制及相关调控因子;对基于PCR的高通量分子筛选方法获取特定环脂肽进行分析,同时对基于调控机制的遗传改造提高假单胞菌产环脂肽的能力和获取更多新型环脂肽等方面的应用进行 展望。     

Detection and characterization of a new beta-conglycinin from soybean seeds

A new protein has been isolated from the reserve proteins of the seeds of soybean (Glycine max) which is particularly deficient in methionine and cysteine. The protein dissociated in sodium dodecyl sulfate into a single polypeptide, M_r 48,000. The amino acid composition, N-terminal leucine and mobility on gel electrophoresis of this polypeptide all were indistinguishable from the β-subunit of β-conglycinin. In its nondissociated form, the protein behaved as a trimer of M_r, 137,000 ± 4000. Its sedimentation coefficient at ionic strength 0.5 was 7.5 S and it possessed antigenic determinants in common with β-conglycinin. This protein therefore has the properties of a new isomer of β-conglycinin—a homogeneous trimer of β subunits.     

Template ability of activated DNA from the regenerating lens

The template ability of DNA changes during the cell cycle. Prior to the initiation of DNA synthesis in the regenerating lens of Triturus, DNA is converted during the latter portion of the G₁ phase into a form which serves as an excellent template for heterologous DNA polymerase. This activated DNA is a much better template than DNA isolated from the G_o phase, or from the early portion of the G₁ phase.     

The template properties of some oligodeoxynucleotides containing cytidine and guanosine

Summary the co-condensation of guanosine- and cytidine-5-phospho-2-methylimidazolide on various oligodeoxynucleotides containing C and G has been studied. We find that GC₇ is an effective template for the incorporation of C into products of the form G_nC, whereas C₇G does not act as a template for C incorporation. The template C₃GC₃GC₃GC₃ directs the synthesis of complementary products, but the yield of long oligomers is very small. Templates in which G residues are contiguous or separated by a single C residue are ineffective, while templates containing the sequence GCCG are very inefficient. The significance of these findings in the context of prebiotic chemistry is discussed.     

Ultrashort cationic lipopeptides and lipopeptoids: Evaluation and mechanistic insights against epithelial cancer cells

Peptides present an attractive scaffold for the development of new anticancer lead agents due to their accessibility and ease of modification. Synthetic ultrashort cationic lipopeptides, with four amino acids or less conjugated to a fatty acid, were developed to retain the biological activity of longer peptides in a smaller molecular size. Herein, we report the activity of amphiphilic lipotripeptides, lipotripeptoids and lipotetrapeptides against breast (MDA-MB-231, JIMT-1), prostate (DU145) and pancreas (MiaPaCa2) epithelial cancer cell lines. The lipotripeptide C16-KKK-NH₂ and lipotetrapeptide C16-P_CatP_HexP_HexP_Cat-NH₂ were identified to possess anticancer activity. The latter lipotetrapeptide possess a short polyproline scaffold consisting of only two L-4R-aminoproline (P_Cat) and two L-4R-hexyloxyproline (P_Hex). However, all the prepared lipotripeptoids lack anticancer activity. The amphiphilic C16-P_CatP_HexP_HexP_Cat-NH₂ exhibited similar anticancer potency to the surfactant benzethonium chloride while superior activity was observed in comparison to myristylamine. Mechanistic studies revealed that the peptides do not lyse ovine erythrocytes nor epithelial cancer cells, thus ruling out necrosis as the mechanism of cell death. Surprisingly, the two lipopeptides exhibit different mechanisms of action that result in cancer cell death. The lipotripeptide C16-KKK-NH₂ was found to induce caspase-mediated apoptosis while C16-P_CatP_HexP_HexP_Cat-NH₂ kills tumor cells independent of caspases.     

Micellar and Biochemical Properties of (Hemi)Fluorinated Surfactants Are Controlled by the Size of the Polar Head

Surfactants with fluorinated and hemifluorinated alkyl chains have yielded encouraging results in terms of membrane protein stability; however, the molecules used hitherto have either been chemically heterogeneous or formed heterogeneous micelles. A new series of surfactants whose polar head size is modulated by the presence of one, two, or three glucose moieties has been synthesized. Analytical ultracentrifugation and small-angle neutron scattering show that fluorinated surfactants whose polar head bears a single glucosyl group form very large cylindrical micelles, whereas those with two or three glucose moieties form small, homogeneous, globular micelles. We studied the homogeneity and stability of the complexes formed between membrane proteins and these surfactants by using bacteriorhodopsin and cytochrome b₆f as models. Homogeneous complexes were obtained only with surfactants that form homogeneous micelles. Surfactants bearing one or two glucose moieties were found to be stabilizing, whereas those with three moieties were destabilizing. Fluorinated and hemifluorinated surfactants with a two-glucose polar head thus appear to be very promising molecules for biochemical applications and structural studies. They were successfully used for cell-free synthesis of the ion channel MscL.     

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。     

Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.