Mechanical properties that influence antimicrobial peptide activity in lipid membranes

Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.     

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces.

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.     

Molecular dynamics investigation of the influence of anionic and zwitterionic interfaces on antimicrobial peptides' structure: implications for peptide toxicity and activity

Molecular dynamics simulations of three related helical antimicrobial peptides have been carried out in zwitterionic diphosphocholine (DPC) micelles and anionic sodiumdodecylsulfate (SDS) micelles. These systems can be considered as model mammalian and bacterial membrane interfaces, respectively. The goal of this study is to dissect the differences in peptide composition which make the mutant peptides (novispirin-G10 and novispirin-T7) less toxic than the parent peptide ovispirin (OVIS), although all three peptides have highly antibacterial properties. Compared to G10 and T7, OVIS inserts deepest into the DPC micelle. This correlates well with the lesser toxicity of G10 and T7. There is strong evidence which suggests that synergistic binding of hydrophobic residues drives binding of OVIS to the micelle. The helical content of G10 and T7 is reduced in the presence of DPC, and this leads to less amphipathic peptide structures, which bind weakly to the micelle. Simulations in SDS were carried out to compare the influence of membrane electrostatics on peptide structure. All three peptides bound strongly to SDS, and retained helical form. This corresponds well with their equally potent antibacterial properties. Based on the simulations, we argue that secondary structure stability often leads to toxic properties. We also propose that G10 and T7 operate by the carpet mechanism of cell lysis. Toxicity of peptides operating by the carpet mechanism can be attenuated by reducing the peptide helical content. The simulations successfully capture experimental binding states, and the different depths of binding of the three peptides to the two micelles correlate with their antibacterial and toxic properties.     

Effect of polymer brush architecture on antibiofouling properties

Polymer brushes show great promise in next-generation antibiofouling surfaces. Here, we have studied the influence of polymer brush architecture on protein resistance. By carefully optimizing reaction conditions, we were able to polymerize oligoglycerol-based brushes with sterically demanding linear or dendronized side chains on gold surfaces. Protein adsorption from serum and plasma was analyzed by surface plasmon resonance. Our findings reveal a pronounced dependence of biofouling on brush architecture. Bulky yet flexible side chains as in dendronized brushes provide an ideal environment to repel protein-possibly through formation of a hydration layer, which can be further enhanced by presenting free hydroxyl groups on the polymer brushes. A deeper understanding of how brush architecture influences protein resistance will ultimately enable fabrication of surface coatings tailored to specific requirements in biomedical applications.     

抗体在二氧化硅表面固定及活性测定

应用分子自组装技术,通过表面羟基化、氨基硅烷化和对苯二甲醛组装,在二氧化硅表面衍生活泼的醛基基团。利用抗体的氨基和醛基发生还原胺化反应将抗体固定在二氧化硅表面,通过抗原抗体反应定性检测固定抗体的生物活性。结果显示应用这种方法能够有效将抗体固定在二氧化硅表面,并保持抗体生物活性,该固定方法在蛋白质检测、分析和蛋白质芯片中有广泛应用价值。     

A Leu-Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

Synthesis and investigation of antimicrobial activity and spectrophotometric and dyeing properties of some novel azo disperse dyes based on naphthalimides

A series of novel disperse dyes containing azo group were synthesized through a diazotization and coupling process. The 4‐amino‐N‐2‐aminomethylpyridine‐1,8‐naphthalimide was diazotized by nitrosylsulphuric acid and coupled with various aromatic amines such as N,N‐diethylaniline, N,N‐dihydroxyethylaniline, 8‐hydroxyquinoline, and 2‐methylindole. Chemical structures of the synthesized dyes were characterized by Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), proton nuclear magnetic resonance (¹H NMR), carbon nuclear magnetic resonance (¹³C NMR), elemental analysis, and ultraviolet–visible (UV–visible) spectroscopy. The spectrophotometric data of all dyes were evaluated in various solvents with different polarity. Eventually, the dyes were applied on polyamide fabrics in order to investigate their dyeing properties. The fastness properties of the dyed fabrics such as wash, light, and rubbing fastness degrees were measured by standard methods. Moreover, the color gamut of the synthesized dyes was measured on polyamide fabrics. Results indicated that some of the synthesized dyes were able to dye polyamide fabrics with deep shades. They had very good wash and rubbing fastness degrees and moderate‐to‐good light fastness on polyamide fabrics. The antibacterial and antifungal activities of the synthesized dyes were evaluated in soluble state and on the dyed fabrics. The results indicated that dye 2 containing N,N‐dihydroxyethylaniline as coupler had the highest activity against all the bacteria and fungi used. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1086–1095, 2015     

Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment

A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.     

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.     

Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are α-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are alpha-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

Functional properties of a novel hybrid antimicrobial peptide NS: potent antitumor activity and efficient plasmid delivery

Antimicrobial peptides have been widely recognized as potential candidates for treating tumor, especially for defending against multidrug‐resistant cells. Previously, based on the structure of substance P, we have designed a novel class of hybrid antimicrobial peptide NS, which possesses potent antimicrobial activity against a broad spectrum of bacterial pathogens. In this study, we evaluated its cytotoxicity to tumor cells and studied the possible mechanism of action. We showed that NS could efficiently kill tumor cells by rapidly disrupting the tumor cell membrane and inhibiting the DNA synthesis. In addition, we also found that NS could efficiently deliver plasmids into cells and exhibit high transfection efficiency after the introduction of a stearyl moiety to its N‐terminus, like many reported cell‐penetrating peptides. Taken together, this study revealed the potential multiple functions of NS, providing fundamental support for further therapeutic application as potential antitumor agent. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

来自穿膜肽的新肽的抗菌活性及抑菌机制

[目的]研究基于穿膜肽和抗菌肽构效关系改造获得的新肽P7的抗菌活性及其对大肠杆菌(E.coli)的抑菌机制.[方法]微量稀释法和溶血实验分析P7的抑菌活性及其对正常细胞的细胞毒性;采用膜荧光探针、流式细胞术和扫描电镜分析P7对E.coli膜通透性、膜完整性的影响和细胞超微结构变化;通过激光共聚焦分析P7在E.coli细胞中的定位;凝胶阻滞实验测定P7与E.coli基因组DNA结合能力.[结果]P7比母肽显示更强的抑菌活性,最低抑菌浓度范围为4-32 μmol/L,且在作用浓度范围内具有较弱的溶血活性.P7可以增加E.coli外膜和内膜的通透性,使E.coli细胞膜的完整性和细胞表面结构受损.同时P7可以穿过E.coli细胞膜在细胞质聚集并与基因组DNA结合.[结论]P7通过增加E.coli内外膜通透性,穿过细胞膜与胞内DNA结合发挥抑菌活性.     

Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH₂) showed the greatest antimicrobial activity (MIC₉₀ of 16 μM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.     

Design of antimicrobial compounds based on peptide structures

New antimicrobial compounds are of major importance because of the growing problem of bacterial resistance and antimicrobial peptides have been gaining a lot of interest. Their mechanism of action, however, is often obscure. Here a set of non-peptidic compounds with antimicrobial activity are presented that have been designed based on criteria derived from three-dimensional structures of antimicrobial peptides. Even though only a small set of compounds has been designed, the activity immediately matches that of the original peptides, supporting the proposed criteria for activity, i.e. not the peptidic nature of antimicrobial peptides is responsible for their activity but rather the proper arrangement of the relevant functional groups.     

The antimicrobial activity of the appetite peptide hormone ghrelin

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.     

New trifluoromethyl quinolone derivatives: Synthesis and investigation of antimicrobial properties

A series of quinolone derivatives, containing different heterocyclic amines were prepared. Synthesized compounds were evaluated for their in vitro antimicrobial activities against two Gram-positive bacteria, three Gram-negative bacteria as well as four fungi. All the derivatives showed good activity towards Gram-positive bacteria and less activity towards Gram-negative bacteria. They also showed moderate to comparable activity against Aspergillus niger and Candida albicans and low to moderate antifungal activity against Aspergillus fumigatus and Aspergillus flavus.