Synergism of Antimicrobial Frog Peptides Couples to Membrane Intrinsic Curvature Strain

Mixtures of the frog peptides magainin 2 and PGLa are well-known for their pronounced synergistic killing of Gram-negative bacteria. We aimed to gain insight into the underlying biophysical mechanism by interrogating the permeabilizing efficacies of the peptides as a function of stored membrane curvature strain. For Gram-negative bacterial-inner-membrane mimics, synergism was only observed when the anionic bilayers exhibited significant negative intrinsic curvatures imposed by monounsaturated phosphatidylethanolamine. In contrast, the peptides and their mixtures did not exhibit significant activities in charge-neutral mammalian mimics, including those with negative curvature, which is consistent with the requirement of charge-mediated peptide binding to the membrane. Our experimental findings are supported by computer simulations showing a significant decrease of the peptide-insertion free energy in membranes upon shifting intrinsic curvatures toward more positive values. The physiological relevance of our model studies is corroborated by a remarkable agreement with the peptide’s synergistic activity in Escherichia coli. We propose that synergism is related to a lowering of a membrane-curvature-strain-mediated free-energy barrier by PGLa that assists membrane insertion of magainin 2, and not by strict pairwise interactions of the two peptides as suggested previously.     

Characterization of the structure and membrane interaction of the antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.     

Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C₁₂EO₈. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C₁₂EO₈. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.     

Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C₁₂EO₈. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C₁₂EO₈. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.     

新疆家蚕抗菌肽抗菌作用的超微结构观察及抗菌机理初探

为探讨基因工程表达的新疆家蚕(Bombyx mori)抗菌肽(cecropin-XJ)的抗菌机制,通过紫外分光光度法研究抗菌肽的抑菌动力学,并采用透射电镜观察抗菌肽作用于金黄色葡萄球菌(Staphylococcus aureus)后的超微结构,对抗菌肽抗菌机理进行初步探讨。结果表明,抗菌肽抑菌作用比较明显,抗菌肽的活性与作用时间有关。抗菌肽可能是通过"桶-板"模式渗透细胞膜,从而影响细胞膜的结构和功能,使细胞膜形成许多孔道,增强了金黄色葡萄球菌细胞的通透性,造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,从而起到抑菌、杀菌作用。抗菌肽使金黄色葡萄球菌细胞内容物大量渗漏而死亡,死亡细胞的细胞壁保持完整,表明细胞膜是抗菌肽作用的主要靶位点。     

Synthetic Antimicrobial Peptides: I. Antimicrobial Activity of Amphiphilic and Nonamphiphilic Cationic Peptides

Comparative antimicrobial properties of three artificial cationic synthetic antimicrobial peptides (SAMP): (RAhaR)₄AhaβA (where R is Arg, Aha is 6-aminohexanoic acid, βA is beta-alanine), (KFF)₃K and R₉F₂ with various amphiphilic properties have been studied relative to pathogenic strains of microorganisms: Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Salmonella enterica, Gram-positive bacteria Staphylococcus aureus, and pathogenic yeast fungus Candida albicans. The selectivity index (SI) values of the peptide preparations were calculated as the ratio of the 50% cytotoxic concentration (TC₅₀) towards eukaryotic host cells to the MIC₅₀ values of the testing antimicrobial peptides. The studied SAMPs appeared to be the most active against the pathogenic yeast fungus C. albicans and the bacterial strains St. aureus and P. aeruginosa. The SI values in these cases exceed 40. Some assumed molecular interactions of the studied SAMPs on the microbial cells have been considered, and possible pathways to increase their antimicrobial activity have been suggested. The proposed SAMPs can serve as a basis for the design and synthesis of new promising synthetic antimicrobial agents.     

生物抗菌肽研究进展

介绍抗菌肽的理化性质、分类、作用机制及其基因工程研究现状和抗菌肽药物开发前景。     

Chemical Composition and Antimicrobial Effect of Western Ethiopian Propolis

Propolis or bee glue is commonly named as a natural resinous mixture produced by honeybees (Apis mellifera) from substances collected from parts of plants, buds, and exudate. The result of the ethyl acetate - methanol (3 : 2) volume by volume fraction yielded a total of two compounds namely betulinic acid and β-amyrin isolated from Bodji Dirmaji and Fincha'a district propolis, respectively. The crude ethanolic extract was portioned with the different solvent systems by increasing the polarities in the following order of hexane, ethyl acetate, and methanol. Column chromatographic method on normal silica gel was used to isolate the compounds. The structures of the compounds were characterized using 1D NMR techniques. The study revealed that western Ethiopian propolis was rich in saponins, tannins, flavonoids, steroids, triterpenes, and glycosides. The antibacterial activity for the isolated compound (betulinic acid) showed the highest inhibition for S. aureus (11.2±1.6), E. coli (17.7±1.1), and A. niger (12.6±1.2) mm.     

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis the solution structure of aurein 1.2.

Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1-3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic alpha-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10-5-10-4 M range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5. 1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.     

两栖动物皮肤结构及皮肤抗菌肽

两栖动物皮肤在自然进化过程中形成了防御病原微生物的三套防御系统,相应地具有特定结构。皮肤抗菌肽是其中先天性防御系统的主要组成部分。本文概述了两栖动物皮肤结构特点以及皮肤抗菌肽在国内外的最新研究进展,重点介绍了两栖动物皮肤腺体和蛙皮抗菌肽的种类、分子结构、抗菌机理、基因表达调控及cDNA编码特点以及基因工程等。以期系统认识和了解这些方面的研究与进展。     

昆虫抗菌肽研究和应用现状

昆虫在受到刺激或感染之后,在其血淋巴中会产生一种抗菌类物质,称抗菌肽。抗菌肽具有分子小、稳定性好、广谱抗菌、无毒副作用等特点,在农业、医药、食品等领域有广泛的应用前景。简要综述了昆虫抗菌肽的基础研究和应用现状。     

枯草芽孢杆菌抗菌肽生物合成的研究进展

革兰氏阳性菌模式生物--枯草芽孢杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质,按合成途径不同,可分为核糖体肽和非核糖体肽。其中,非核糖体肽分子量较小,一般为3000Da以下,其生物合成是通过多功能复合酶系--非核糖体肽链合成酶来完成的,多发生在菌体生长停止之后;而核糖体肽分子量较大,其合成多于菌体快速生长时期。非核糖体肽链合成酶和核糖体肽的合成及其调控均需基因参与,而这一系列基因就构成了各种抗菌肽生物合成的基因簇。对核糖体肽和非核糖体肽的生物合成及其相关调控机制进行了综述。     

Synthetic Antimicrobial Peptides. II. Antimicrobial and Hemolytic Activity of Cationic Peptides Containing Cysteine Residues with Free Sulfhydryl Groups

Russian Journal of Bioorganic Chemistry - The antimicrobial and hemolytic activities of R9F2С2 (P1ss), (KFF)3KС2 (P2ss), and (RAhaR)4AhaβAС2 (P3ss) (where Aha is...     

Inhibition of KCa2.2 and KCa2.3 channel currents by protonation of outer pore histidine residues

Ion channels are often modulated by changes in extracellular pH, with most examples resulting from shifts in the ionization state of histidine residue(s) in the channel pore. The application of acidic extracellular solution inhibited expressed K_Ca2.2 (SK2) and K_Ca2.3 (SK3) channel currents, with K_Ca2.3 (pIC₅₀ of ∼6.8) being approximately fourfold more sensitive than K_Ca2.2 (pIC₅₀ of ∼6.2). Inhibition was found to be voltage dependent, resulting from a shift in the affinity for the rectifying intracellular divalent cation(s) at the inner mouth of the selectivity filter. The inhibition by extracellular protons resulted from a reduction in the single-channel conductance, without significant changes in open-state kinetics or open probability. K_Ca2.2 and K_Ca2.3 subunits both possess a histidine residue in their outer pore region between the transmembrane S5 segment and the pore helix, with K_Ca2.3 also exhibiting an additional histidine residue between the selectivity filter and S6. Mutagenesis revealed that the outer pore histidine common to both channels was critical for inhibition. The greater sensitivity of K_Ca2.3 currents to protons arose from the additional histidine residue in the pore, which was more proximal to the conduction pathway and in the electrostatic vicinity of the ion conduction pathway. The decrease of channel conductance by extracellular protons was mimicked by mutation of the outer pore histidine in K_Ca2.2 to an asparagine residue. These data suggest that local interactions involving the outer turret histidine residues are crucial to enable high conductance openings, with protonation inhibiting current by changing pore shape.     

The Effect of Membrane Lipid Composition on the Formation of Lipid Ultrananodomains

Some lipid mixtures form membranes containing submicroscopic (nanodomain) ordered lipid domains (rafts). Some of these nanodomains are so small (radius <5 nm) that they cannot be readily detected with Förster resonance energy transfer (FRET)-labeled lipid pairs with large Ro. We define such domains as ultrananodomains. We studied the effect of lipid structure/composition on the formation of ultrananodomains in lipid vesicles using a dual-FRET-pair approach in which only one FRET pair had Ro values that were sufficiently small to detect the ultrananodomains. Using this approach, we measured the temperature dependence of domain and ultrananodomain formation for vesicles composed of various mixtures containing a high-T_m lipid (brain sphingomyelin (SM)) or dipalmitoyl phosphatidylcholine (DPPC)), low-T_m lipid (dioleoylphosphatidylcholine (DOPC) or 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC)), and a lower (28 mol %) or higher (38 mol %) cholesterol concentration. For every lipid combination tested, the thermal stabilities of the ordered domains were similar, in agreement with our prior studies. However, the range of temperatures over which ultrananodomains formed was highly lipid-type dependent. Overall, vesicles that were closest to mammalian plasma membrane in lipid composition (i.e., with brain SM, POPC, and/or higher cholesterol) formed ultrananodomains in preference to larger domains over the widest temperature range. Relative to DPPC, the favorable effect of SM on ultrananodomain formation versus larger domains was especially large. In addition, the favorable effect of a high cholesterol concentration, and of POPC versus DOPC, on the formation of ultrananodomains versus larger domains was greater in vesicles containing SM than in those containing DPPC. We speculate that it is likely that natural mammalian lipids are tuned to maximize the tendency to form ultrananodomains relative to larger domains. The observation that domain size is more sensitive than domain formation to membrane composition has implications for how membrane domain properties may be regulated in vivo.     

一类蛙源非典型结构抗菌肽cDNA的克隆以及成熟肽的预测

抗菌肽是两栖类非特异性免疫的重要组成部分,具有广谱的抗菌、抗病毒、抗肿瘤、抗寄生虫等活性,而且不易产生耐药性.抗菌肽的功能发挥与其特有的α螺旋结构密切相关,但是一些抗菌肽在水溶液中呈无规则卷曲,在类膜溶液中才转变成α螺旋,这类抗菌肽往往表现出极强的抗菌肽活性或细胞毒性,在药物开发中提供更多的利用潜能.本文从东北林蛙(Rana dybowskii)皮肤组织中,通过RT-PCR技术,克隆了一类无规则卷曲的抗菌肽,属于chensirin-2家族.19条不同的抗菌肽的cDNA序列共编码3种长度为14个氨基酸残基的成熟肽,分子量在1450.78-1460.82之间,理论等电点在9.53-9.70之间,3种抗菌肽都有两亲性和阳离子性,二级结构呈现无规卷曲.这些理化性质预示着这3种抗菌肽可能具有特殊的药用价值.     

L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection.     

Antimicrobial Activity of Selected Antimicrobial Peptides Against Planktonic Culture and Biofilm of Acinetobacter baumannii

Probiotics and Antimicrobial Proteins - Acinetobacter baumannii is one of the most challenging pathogens, on account of its predisposition to develop resistance leading to severe,...