Design of Gram-negative selective antimicrobial peptides

Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, signals bacterial invasion and triggers defensive host responses. However, excessive responses also lead to the serious pathophysiological consequence of septic shock. To develop Gram-negative selective compounds that can inhibit the effects of LPS-induced sepsis, we have designed constrained cyclic antimicrobial peptides based on a cystine-stabilized beta-stranded framework mimicking the putative LPS-binding sites of the LPS-binding protein family. Our prototype termed R4A, c(PACRCRAG-PARCRCAG), consists of an eight amino acid degenerated repeat constrained by a head-to-tail cyclic peptide backbone and two cross-bracing disulfides. NMR study of K4A, an R4A analogue with four Arg --> Lys replacements, confirmed the amphipathic design elements with four Lys on one face of the antiparallel beta-strand and two hydrophobic cystine pairs plus two Ala on the opposite face. K4A and R4A displayed moderate microbicidal potency and Gram-negative selectivity. However, R4A analogues with single or multiple replacements of Ala and Gly with Arg or bulky hydrophobic amino acids displayed increased potency and selectivity in both low- and high-salt conditions. Analogues R5L and R6Y containing additional cationic and bulky hydrophobic amino acids proved the best mimics of the amphipathic topology of the "active-site" beta-strands of LPS-binding proteins. They displayed potent activity against Gram-negative E. coli with a minimal inhibitory concentration of 20 nM and a >200-fold selectivity over Gram-positive S. aureus. Our results suggest that an LPS-targeted design may present an effective approach for preparing selective peptide antibiotics.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

The bacterial surface layer provides protection against antimicrobial peptides

This report describes a previously unrecognized role for bacterial surface layers as barriers that confer protection against antimicrobial peptides. As antimicrobial peptides exist in natural environments, S-layers may provide a bacterial survival mechanism that has been selected for through evolution.     

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B

A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein’s C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.     

Branched antimicrobial peptides

Branched peptides E(RLAR)2, E[E(RLAR)2]2, E(KLAR)2, and E[E(KLAR)2]2 were synthesized on the basis of tetrapeptides RLAR and KLAR and glutamic acid bis(pentafluorophenyl) ester. Their minimal antimicrobial concentrations were shown to decrease along with increase in branching, achieving 12 microM for Escherichia coli cells, which is comparable to antimicrobial activities of temporin, magainin, and dermaseptin. The branched peptides were found not to act on human erythrocytes.     

Branched antimicrobial peptides

Branched peptides E(RLAR)₂, E[E(RLAR)₂]₂, and E(KLAR)₂, E[E(KLAR)₂]₂ were synthesized on the basis of tetrapeptides RLAR and KLAR and glutamic acid bis(pentafluorophenyl) ester. Their minimal antimicrobial concentrations were shown to decrease along with increase in branching, achieving 12 μM for Escherichia coli cells, which is comparable to antimicrobial activities of temporin, magainin, and dermaseptin. The branched peptides were found not to act on human erythrocytes.     

Plant antimicrobial peptides

Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.     

Design of salt-insensitive glycine-rich antimicrobial peptides with cyclic tricystine structures

Cyclic peptide backbone and cystine constraints were used to develop a broadly active salt-insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eight Gly residues in an 18-residue sequence. The importance of rigidity and amphipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobial peptide. The first series, which retained the charge and hydrophobic amino acids of tachyplesin, but contained zero to four covalent constraints, included a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] analogues were prepared in a parallel series with all six bulky hydrophobic amino acids in their sequences replaced with Gly. Circular dichroism measurements showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sheet structures, while the less constrained [Gly(6)] analogues were disordered. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad activity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large variations in activity spectra and significant decreases in potency that roughly correlated with the decreases in conformational constraints. Except against Escherichia coli, the Gly-rich analogues with two or fewer covalent constraints were largely inactive under high-salt conditions. Remarkably, the most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum against all 10 test microbes in both low- and high-salt assays. Collectively, our results show that [Gly(6)]ccTP 1acould serve as a template for further analogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.     

抗菌肽活性及天然抗菌肽的改造

抗菌肽具有抗菌谱广、热稳定性强、分子量小及免疫原性小等特点,其杀菌机制独特,病原菌不易产生耐药性,有望开发成新一代肽类抗生素。本文主要综述了影响抗菌肽生物活性的生化性质,即螺旋度、疏水性、两亲性、正电荷数等,并从结构的角度论述了其对抗菌肽抑菌活性的影响。部分抗菌肽具有空间结构不稳定、溶血活性等缺点,限制了其临床应用。因此,对天然抗菌肽的改造也成为目前抗菌肽的研究热点,本文还综述了天然抗菌肽的改造方法。     

Design of model amphipathic peptides having potent antimicrobial activities.

Induced amphipathic alpha-helical conformations play an important role in the biological activity of peptides. By using reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence (Ac-LKLLKKLLKKLKKLLKKL-NH2) was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC. This peptide exhibited potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. We have prepared a complete set of omission, as well as of leucine and lysine substitution, analogs of this sequence. These analogs were used to investigate the effects of such alterations on the parent sequence's antimicrobial and hemolytic activities relative to each analog's behavior during RP-HPLC. The potential for the formation of ion channels through cell membranes by this amphipathic model peptide was also evaluated through preparation of analogs which varied in length from 8 to 22 residues, while maintaining their amphipathicity.     

Defensins: antimicrobial peptides for therapeutic development

A novel class of endogenous antimicrobial peptides called defensins has shown great versatility in their activity against a diverse range of microorganisms including bacteria, viruses and fungi. Their mode of action of bacterial cell lysis seems largely nonspecific and so promises to avert the development of resistance. These two features have made them an area of intense research activity and growing commercial interest. A successful multidisciplinary effort to investigate and develop novel defensins analogues has been established in Singapore that involves computer modeling, biochemistry, proteomics, chemical synthesis, molecular biology and clinical sciences.     

Leishmania donovani: Thionins, plant antimicrobial peptides with leishmanicidal activity

The leishmanicidal activity of plant antibiotic peptides (PAPs) from the principal families, such wheat thionins, a barley lipid transfer protein and potato defensins and snakins were tested in vitro against Leishmania donovani. Only thionins and defensins were active against this human pathogen at a low micromolar range of concentrations. Thionins resulted as the most active peptides tested until now. They collapsed ionic and pH gradients across the parasite plasma membrane together with a rapid depletion of intracellular ATP without affecting mitochondrial potential. Hence the lethal effect of thionins was mostly associated to permeabilization of the plasma membrane leading to an immediate death of the parasite. The present work is the first evidence for leishmanicidal activity in plant peptides. Future prospects for their development as new antiparasite agents on human diseases are considered.     

Computational resources and tools for antimicrobial peptides

Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immune system, protect against pathogens including bacteria, fungi, viruses, and parasites. In general, AMPs are relatively small peptides (<10 kDa) with cationic nature and amphipathic structure and have modes of action different from traditional antibiotics. Up to now, there are more than 19 000 AMPs that have been reported, including those isolated from nature sources or by synthesis. They have been considered to be promising substitutes of conventional antibiotics in the quest to address the increasing occurrence of antibiotic resistance. However, most AMPs have modest direct antimicrobial activity, and their mechanisms of action, as well as their structure–activity relationships, are still poorly understood. Computational strategies are invaluable assets to provide insight into the activity of AMPs and thus exploit their potential as a new generation of antimicrobials. This article reviews the advances of AMP databases and computational tools for the prediction and design of new active AMPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

抗菌肽的研究进展

近年来,由于细菌耐药性问题日趋严峻,开发新型抗菌制剂已迫在眉睫。抗菌肽具有相对分,子质量小、对热稳定、抗菌谱广及不同于抗生素的抗菌机制,不产生耐药性,因而具有重要的临床应用价值。本文对天然来源、蛋白质酶解、化学合成及基因工程方法产生的抗菌肽及其研究进展进行了综述。     

Lactococcal membrane-permeabilizing antimicrobial peptides

A number of lactococcal antimicrobial peptides, bacteriocins have been discovered and characterized. Since Lactococcus spp. are generally regarded as safe bacteria, their bacteriocins are expected for various application uses. Most of lactococcal bacteriocins exert antimicrobial activity via membrane permeabilization. The most studied and prominent bacteriocin, nisin A is characterized in the high activity and has been utilized as food preservatives for more than half a century. Recently, other lactococcal bacteriocins such as lacticin Q were found to have distinguished features for further applications as the next generation to nisin.     

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Expression systems for heterologous production of antimicrobial peptides

Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.