Amphipathic alpha helical antimicrobial peptides.

Antimicrobial peptides (AMPs) that assume an amphipathic alpha helical structure are widespread in nature. Their activity depends on several parameters including the sequence, size, degree of structure formation, cationicity, hydrophobicity and amphipathicity. The analysis of numerous natural AMPs provided representative values for these parameters and led to a sequence template with which to generate potent artificial lead AMPs. Sequences were then varied in a rational manner, using both natural and nonproteinogenic amino acids, to probe the individual roles of each parameter in modulating biological activity. A high cationicity combined with a stabilized amphipathic alpha helical structure conferred enhanced cidal activity towards all the cell types considered, and was a requirement for Gram-positive bacteria and fungi. An elevated helicity also correlated with increased hemolytic activity. The structural requirements for activity against several Gram-negative bacteria were instead considerably less stringent, so that it persisted in peptides in which formation of a helical structure and/or amphipathicity were impeded. Either a reduced charge or a reduced hydrophobicity resulted in generally inactive peptides. These observations, combined with the kinetics of bacterial membrane permeabilization and time-killing are discussed in terms of currently accepted models of action for this type of peptide. The simple guidelines obtained in this study allowed the design of highly active shortened AMPs and may be generally useful in the development of this type of peptides as anti-infective agents.     

Biotic stress resistance in agriculture through antimicrobial peptides

Antimicrobial peptides (AMPs) are the hosts' defense molecules against microbial pathogens and gaining extensive research attention worldwide. These have been reported to play vital role of host innate immunity in response to microbial challenges. AMPs can be used as a natural antibiotic as an alternative of their chemical counterpart for protection of plants/animals against diseases. There are a number of sources of AMPs including prokaryotic and eukaryotic organisms and are present, both in vertebrates and invertebrates. AMPs can be classified as cationic or anionic, based on net charges. Large number of databases and tools are available in the public domain which can be used for development of new genetically modified disease resistant varieties/breeds for agricultural production. The results of the biotechnological research as well as genetic engineering related to AMPs have shown high potential for reduction of economic losses of agricultural produce due to pathogens. In this article, an attempt has been made to introduce the role of AMPs in relation to plants and animals. Their functional and structural characteristics have been described in terms of its role in agriculture. Different sources of AMPs and importance of these sources has been reviewed in terms of its availability. This article also reviews the bioinformatics resources including different database tools and algorithms available in public domain. References of promising biotechnology research in relation to AMPs, prospects of AMPs for further development of genetically modified varieties/breeds are highlighted. AMPs are valuable resource for students, researchers, educators and medical and industrial personnel.     

Staphylococcal resistance to antimicrobial peptides of mammalian and bacterial origin

Antimicrobial host defense peptides, such as defensins, protegrins, and platelet microbicidal proteins are deployed by mammalian skin, epithelia, phagocytes, and platelets in response to Staphylococcus aureus infection. In addition, staphylococcal products with similar structures and activities, called bacteriocins, inhibit competing microorganisms. Staphylococci have developed resistance mechanisms, which are either highly specific for certain host defense peptides or bacteriocins or which broadly protect against a range of cationic antimicrobial peptides. Experimental infection models can be used to study the molecular mechanisms of antimicrobial peptides, the peptide resistance strategies of S. aureus, and the therapeutic potential of peptides in staphylococcal diseases.     

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.     

The co-evolution of host cationic antimicrobial peptides and microbial resistance

Endogenous cationic antimicrobial peptides (CAMPs) are among the most ancient and efficient components of host defence. It is somewhat of an enigma that bacteria have not developed highly effective CAMP-resistance mechanisms, such as those that inhibit many therapeutic antibiotics. Here, we propose that CAMPs and CAMP-resistance mechanisms have co-evolved, leading to a transient host-pathogen balance that has shaped the existing CAMP repertoire. Elucidating the underlying principles of this process could help in the development of more sustainable antibiotics.     

Effect of salt on the interactions of antimicrobial peptides with zwitterionic lipid bilayers

The effect of salt on the binding of the antimicrobial peptide magainin to POPC lipid bilayers is studied by 40-50 ns molecular dynamics simulations of a POPC bilayer in the presence of different concentrations of Na+ and Cl- ions, corresponding to effective concentrations of 0, 100, 150, 200, 250 and 300 millimolar NaCl, with and without a single molecule of antimicrobial peptide magainin. Simulations without magainin showed that increasing salt concentration leads to the decrease in the area per lipid, a decrease in the head group tilt of the lipids, as well as increased order of lipid tails, in agreement with other recent simulations. Simulations with magainin show that peptide binding to the lipids is stronger at lower concentrations of salt. The peptides disorder the lipids in their immediate vicinity, but this effect is diminished as the salt concentration increases. Our studies indicate that while 50 ns simulations give information on peptide hydrogen bonding and lipid tail ordering that is insensitive to the initial peptide orientation, this run time is not sufficient to equilibrate the peptide position and orientation within the bilayer.     

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.     

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.     

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.     

Cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance

Cathelicidins are a gene family of antimicrobial peptides produced as inactive precursors. Signal peptidase removes the N-terminal signal sequence, while peptidylglycine alpha-amidating monooxygenase often amidates and cleaves the C-terminal region. Removal of the cathelin domain liberates the active antimicrobial peptide. For mammalian sequences, this cleavage usually occurs through the action of elastase, but other tissue-specific processing enzymes may also operate. Once released, these bioactive peptides are susceptible to proteolytic degradation. We propose that some mature cathelicidins are naturally resistant to proteases due to their unusual primary structures. Among mammalian cathelicidins, proline-rich sequences should resist attack by serine proteases because proline prevents cleavage of the scissile bond. In hagfish cathelicidins, the unusual amino acid bromotryptophan may make the active peptides less susceptible to proteolysis for steric reasons. Such protease resistance could extend the pharmacokinetic lifetimes of cathelicidins in vivo, sustaining antimicrobial activity.     

A mammalian lysosomal membrane protein confers multidrug resistance upon expression in Saccharomyces cerevisiae

Mouse transporter protein (MTP) is a highly conserved polytopic membrane protein present in mammalian lysosomes and endosomes. The role of MTP in regulating the in vivo subcellular distribution of numerous structurally distinct small molecules has been examined in this study by its expression in a drug-sensitive strain of the yeast Saccharomyces cerevisiae. Surprisingly, the expression of MTP in membranes of an intracellular compartment resulted in a cellular resistance or hypersensitivity to a range of drugs that included nucleoside and nucleobase analogs, antibiotics, anthracyclines, ionophores, and steroid hormones. The intracellular bioavailability of steroid hormones was altered by MTP, as determined using an in vivo glucocorticoid receptor-driven reporter assay in yeast, suggesting that the MTP-regulated drug sensitivity arose due to a change in the subcellular compartmentalization of steroid hormones and other drugs. MTP-regulated drug sensitivity in yeast was blocked to varying degrees by compounds that inhibit lysosomal function, interfere with intracellular cholesterol transport, or modulate the multidrug resistance phenotype of mammalian cells. These results indicate that MTP is involved in the subcellular compartmentalization of diverse hydrophobic small molecules and contributes to the inherent drug sensitivity or resistance of the mammalian cell.     

Origin of low mammalian cell toxicity in a class of highly active antimicrobial amphipathic helical peptides

We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316-12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.     

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。     

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。     

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.