The rothein peptides from the skin secretion of Roth's tree frog Litoria rothii. Sequence determination using positive and negative ion electrospray mass spectrometry

The secretion from the dorsal glands of the frog Litoria rothii contains a series of new peptides including rothein 1 (SVSNIPESIGF-OH, a neuropeptide which contracts smooth muscle), a number of inactive rothein 2 and 3 peptides (e.g. rothein 2.1, AGGLDDLLEPVLNSADNLVHGL-OH), and a new proline rich peptide, named rothein 4.1 (AEILFGDVRPPWMPPPIFPEMP-OH), which shows neither antimicrobial nor neuronal nitric oxide synthase (nNOS) activity. Two known neuropeptides of the caerulein family [e.g. caerulein, pEQDY(SO3)TGWMDF-NH2] together with a series of known caerin 1 antibiotic and nNOS-inhibiting peptides (e.g. caerin 1.1, GLLSVLGSVAKHVLPHVVPVIAEHL-NH2) were also identified. Positive ion electrospray mass spectrometry (ES-MS) was used as the primary method to investigate the sequences of the new peptides. Negative ion ES-MS was used to fill in any gaps in the positive ion data and, finally, Edman automated sequencing was used to differentiate between Leu and Ile and to confirm the sequences determined by mass spectrometry.     

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis. Part 2. Sequence determination using electrospray mass spectrometry

Sixteen aurein peptides are present in the host defence secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aureus and seventeen from those of the related Southern Bell Frog Litoria raniformis. All peptides have been sequenced using a combination of electrospray mass spectrometry and Lys-C digestion, with each sequence confirmed by automated Edman sequencing. The peptides are named in five groups, viz. aureins 1-5. Ten of these peptides are common to both species of frog. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), the smallest peptide from an anuran reported to have both antibiotic and anticancer activity; aurein 2.2 (GLLDIVKKVIGAFGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). The aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5.1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH), show neither antibacterial nor anticancer activity.     

HPLC and MALDI investigation of the stress influence on the composition of skin secretion of the Common frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Skin secretory amphibian antimicrobial peptides are the part of their immune defense. The present work is devoted to the study of the influence of “water environment stress” and additional bacterial impact on the composition of the skin secretion of the Common frog (Rana temporaria) by means of high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry. It was shown that the contact of the amphibian species with Micrococcus luteus and Staphylococcus aureus stimulates the release of antimicrobial peptides, maintains the high bradykinin and related peptides levels in the skin secretion and influences the processing of the latter ones. The possibilities of mass spectrometric profiling by using HPLC and MALDI were demonstrated. This feature allows the detection of potentially bioactive peptides for their future direct testing, as has been shown for temporin M and brevinin 1Tb in the present study.     

Dendropsophin 1, a novel antimicrobial peptide from the skin secretion of the endemic Colombian frog Dendropsophus columbianus

In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher α-helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.     

Membrane Permeabilization and Antimicrobial Activity of Recombinant Defensin-d2 and Actifensin against Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans

Antimicrobial resistance requires urgent efforts towards the discovery of active antimicrobials, and the development of strategies to sustainably produce them. Defensin and defensin-like antimicrobial peptides (AMPs) are increasingly gaining pharmacological interest because of their potency against pathogens. In this study, we expressed two AMPs: defensin-d2 derived from spinach, and defensin-like actifensin from Actinomyces ruminicola. Recombinant pTXB1 plasmids carrying the target genes encoding defensin-d2 and actifensin were generated by the MEGAWHOP cloning strategy. Each AMP was first expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and was thereafter assayed for antimicrobial activity against multidrug-resistant (MDR) pathogens. Approximately 985 µg/mL and 2895 µg/mL of recombinant defensin-d2 and actifensin, respectively, were recovered with high purity. An analysis by MALDI-TOF MS showed distinct peaks corresponding to molecular weights of approximately 4.1 kDa for actifensin and 5.8 kDa for defensin-d2. An in vitro antimicrobial assay showed that MDR Pseudomonas aeruginosa and Candida albicans were inhibited at minimum concentrations of 7.5 µg/mL and 23 µg/mL for recombinant defensin-d2 and actifensin, respectively. The inhibitory kinetics of the peptides revealed cidal activity within 4 h of the contact time. Furthermore, both peptides exhibited an antagonistic interaction, which could be attributed to their affinities for similar ligands, as deduced by peptide–ligand profiling. Moreover, both peptides inhibited biofilm formation, and they exhibited no resistance potential and low hemolytic activity. The peptides also possess the ability to permeate and disrupt the cell membranes of MDR P. aeruginosa and C. albicans. Therefore, recombinant actifensin and defensin-d2 exhibit broad-spectrum antimicrobial activity and have the potential to be used as therapy against MDR pathogens.     

Peptide interactions with bacterial lipopolysaccharides

Peptide and protein interactions with (lipo)polysaccharides are important in various biological contexts, including lipoprotein deposition at proteoglycan-covered endothelial surfaces in atherosclerosis, lectin functionality, and the interaction of antimicrobial and anti-inflammatory peptides and proteins with (lipo)polysaccharides. The latter of these areas, which is the topic of this review, has attracted considerable interest during the last few years, since antimicrobial peptides may offer novel therapeutic opportunities in an era of growing problems with antibiotic resistance, and persisting problems with both acute and chronic inflammation. In the present overview, physicochemical factors affecting peptide interactions with bacterial (lipo)polysaccharides are discussed, both in solution and at membrane interfaces. In doing so, an attempt is made to illustrate how physicochemical factors affect the antimicrobial and anti-endotoxic functionality of such peptides, and how knowledge on this can be translated into therapeutic opportunities, e.g., in sepsis.     

Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues

Antimicrobial peptides (AMPs) are effector molecules of innate immune systems found in different groups of organisms, including microorganisms, plants, insects, amphibians and humans. These peptides exhibit several structural motifs but the most abundant AMPs assume an amphipathic alpha-helical structure. The alpha-helix forming antimicrobial peptides are excellent candidates for protein engineering leading to an optimization of their biological activity and target specificity. Nowadays several approaches are available and this review deals with the use of combinatorial synthesis and directed evolution in order to provide a high-throughput source of antimicrobial peptides analogues with enhanced lytic activity and specificity.     

Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics

Antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. There is currently an extensive effort to understand the mode of action of antimicrobial peptides which are considered as one alternative to classical antibiotics. The main advantage of this class of substances, when considering bacterial resistance, is that they rapidly, within minutes, kill bacteria. Antimicrobial peptides can be found in every organism and display a wide spectrum of activity. Hence, the goal is to engineer peptides with an improved therapeutic index, i.e. high efficacy and target specificity. For the rational design of such novel antibiotics it is essential to elucidate the molecular mechanism of action. Biophysical studies have been performed using to a large extent membrane model systems demonstrating that there are distinctive different mechanisms of bacterial killing by antimicrobial peptides. One can distinguish between peptides that permeabilize and/or disrupt the bacterial cell membrane and peptides that translocate through the cell membrane and interact with a cytosolic target. Lantibiotics exhibit specific mechanisms, e.g. binding to lipid II, a precursor of the peptidoglycan layer, either resulting in membrane rupture by pore formation or preventing cell wall biosynthesis. The classical models of membrane perturbation, pore formation and carpet mechanism, are discussed and related to other mechanisms that may lead to membrane dysfunction such as formation of lipid-peptide domains or membrane disruption by formation of non-lamellar phases. Emphasis is on the role of membrane lipid composition in these processes and in the translocation of antimicrobial peptides.     

Host defense peptides and lipopeptides: modes of action and potential candidates for the treatment of bacterial and fungal infections

Endogenous peptide antibiotics (termed also host-defense or antimicrobial peptides) are known as evolutionarily old components of innate immunity. They were found initially in invertebrates, but later on also in vertebrates, including humans. This secondary, chemical immune system provides organisms with a repertoire of small peptides that act against invasion (for both offensive and defensive purposes) by occasional and obligate pathogens. Each antimicrobial peptide has a broad but not identical spectrum of antimicrobial activity, predominantly against bacteria, providing the host maximum coverage against a rather broad spectrum of microbial organisms. Many of these peptides interact with the target cell membranes and increase their permeability, which results in cell lysis. A second important family includes lipopeptides. They are produced in bacteria and fungi during cultivation on various carbon sources, and possess a strong antifungal activity. Unfortunately, native lipopeptides are non-cell selective and therefore extremely toxic to mammalian cells. Whereas extensive studies have emerged on the requirements for a peptide to be antibacterial, very little is known concerning the parameters that contribute to antifungal activity. This review summarizes recent studies aimed to understand how antimicrobial peptides and lipopeptides select their target cell. This includes a new group of lipopeptides highly potent against pathogenic fungi and yeast. They are composed of inert cationic peptides conjugated to aliphatic acids with different lengths. Deep understanding of the molecular mechanisms underlying the differential cells specificity of these families of host defense molecule is required to meet the challenges imposed by the life-threatening infections.     

Host-defence skin peptides of the Australian Streambank Froglet Crinia riparia: isolation and sequence determination by positive and negative ion electrospray mass spectrometry

A combination of positive and negative ion electrospray mass spectrometry (ES-MS) together with automated Edman sequencing has been used to determine the amino acid sequences of the host-defence peptides from the skin glands of the froglet Crinia riparia. The peptides are called riparins. Of the eight peptides isolated, five are neuropeptides containing intramolecular disulfide linkages; e.g. the major peptide riparin 1.4 (FFLPPCAYKGTC-OH). Positive ion ES-MS identifies the five residues of riparin 1.4 outside the disulfide moiety, but provides no information on the sequence within the disulfide ring. In contrast, the negative ion dissociations of the [M-H]- ion of riparin 1.4 identify the --S-S-- link by loss of H2S2 from the [M-H]- ion, and also provide the sequence within the disulfide unit. Other peptides are riparin 2.1 [(IIEKLVNTALGLLSGL-NH2), a narrow-spectrum antibiotic], signiferin 3.1 [(GIAEFLNYIKSKA-NH2), an nNOS inhibitor] and riparin 5.1 [IVSYPDDAGEHAHKMG-NH2], which shows no neuropeptide, antibiotic or nNOS activity.     

The fallaxidin peptides from the skin secretion of the Eastern Dwarf Tree Frog Litoria fallax. Sequence determination by positive and negative ion electrospray mass spectrometry: antimicrobial activity and cDNA cloning of the fallaxidins

The glandular skin secretion of the Eastern Dwarf Tree Frog Litoria fallax contains nine peptides named fallaxidins. The sequences of these peptides were elucidated using a combination of positive and negative electrospray mass spectrometry together with Edman sequencing. Among these peptides are: (i) fallaxidins 1.1 and 2.1 which have the sequences YFPIPI-NH2 and FWPFM-NH2. The activities of these peptides are unknown, but it has been shown that they are not smooth muscle active, opioids or antimicrobially active, nor do they effect proliferation of lymphocytes; (ii) two weakly active antibiotics, fallaxidins 3.1 and 3.2 (e.g. fallaxidin 3.1, GLLDLAKHVIGIASKL-NH2), and a moderately active antibiotic fallaxidin 4.1 (GLLSFLPKVIGVIGHLIHPPS-OH). Fallaxidin 4.1 has an unusual sequence for an antibiotic, containing three Pro residues together with a C-terminal CO2H group. cDNA cloning has confirmed the identity of the nine isolated peptides from L. fallax, together with five additional peptides not detected in the peptide profile. The pre-regions of the nine preprofallaxidins are conserved and similar to those of the caerin peptides from L. caerulea and L. splendida, suggesting that the fallaxidin and caerin peptides, although significantly different in sequence, originated from a common ancestor gene.     

Using intrinsic X-ray absorption spectral differences to identify and map peptides and proteins

The intrinsic variation in the near-edge X-ray absorption fine structure (NEXAFS) spectra of peptides and proteins provide an opportunity to identify and map them in various biological environments, without additional labeling. In principle, with sufficiently accurate spectra, peptides (<50 amino acids) or proteins with unusual sequences (e.g., cysteine- or methionine-rich) should be differentiable from other proteins, since the NEXAFS spectrum of each amino acid is distinct. To evaluate the potential for this approach, we have developed X-SpecSim, a tool for quantitatively predicting the C, N, and O 1s NEXAFS spectra of peptides and proteins from their sequences. Here we present the methodology for predicting such spectra, along with tests of its precision using comparisons to the spectra of various proteins and peptides. The C 1s, N 1s, and O 1s spectra of two novel antimicrobial peptides, Indolicidin (ILPWKWPWWPWRR-NH2) and Sub6 (RWWKIWVIRWWR-NH2), as well as human serum albumin and fibrinogen are reported and interpreted. The ability to identify, differentiate, and quantitatively map an antimicrobial peptide against a background of protein is demonstrated by a scanning transmission X-ray microscopy study of a mixture of albumin and sub6.     

Antimicrobial activity and protease stability of peptides containing fluorinated amino acids

Selective fluorination of peptides results in increased chemical and thermal stability with simultaneously enhanced hydrophobicity. We demonstrate here that fluorinated derivatives of two host defense antimicrobial peptides, buforin and magainin, display moderately better protease stability while retaining, or exhibiting significantly increased bacteriostatic activity. Four fluorinated analogues in the buforin and two in the magainin series were prepared and analyzed for (1) their ability to resist hydrolytic cleavage by trypsin; (2) their antimicrobial activity against both gram-positive and gram-negative bacterial strains; and (3) their hemolytic activity. All but one fluorinated peptide (M2F5) showed retention, or significant enhancement, of antimicrobial activity. The peptides also showed modest increases in protease resistance, relative to the parent peptides. Only one of the six fluorinated peptides (BII1F2) was degraded by trypsin at a slightly faster rate than the parent peptide. Hemolytic activity of peptides in the buforin series was essentially null, while fluorinated magainin analogues displayed an increase in hemolysis compared to the parent peptides. These results suggest that fluorination may be an effective strategy to increase the stability of biologically active peptides where proteolytic degradation limits therapeutic value.     

Lantibiotics as surface active agents for biomedical applications

The unique physical structure of lantibiotics, (e.g. double bonds, thioethers rings, and unusual amino acid residues), makes these antimicrobial peptides highly reactive, and thus different in mode-of-action from clinical antibiotics. Members of the lantibiotic group have been successfully tested against pathogenic organisms for decades. Some lantibiotics have been studied for use in treating skin infections, and several have been characterized for their anti-viral activity. In addition to their antimicrobial capabilities, lantibiotics possess amphiphilic characteristics, making them potentially valuable as emulsifiers in drug formulations. The small size and surface activity of these peptides also may allow them to enhance the transport of therapeutic compounds across cell membranes. Over 30 lantibiotics are presently known, and more are being discovered each year. With the rising incidence of resistant bacteria, lantibiotics offer considerable potential as safe, antimicrobial barriers for use on synthetic material surfaces, as emulsifiers in formulation of hydrophobic drugs, and as absorption promoters for selected compounds across mucosal membranes. The major hindrance to research in this area is that lantibiotics are difficult to obtain. But with improved methods for production and purification of these unusual antimicrobial agents, the promise of cost-effective application of lantibiotics may eventually be realized.     

Bacterial lantibiotics: strategies to improve therapeutic potential

Lantibiotics are ribosomally-synthesised antimicrobial peptides produced by Gram-positive bacteria that are characterised by the presence of lanthionine and/or methyllanthionine residues. Other unusual post-translationally modified amino acids, most frequently dehydroalanine and dehydrobutyrine, can also be present. While it has been frequently suggested that these peptides have the potential to be utilised in a wide range of medical applications, to date no actual therapeutic applications have been convincingly described. More recently, however, they have been the focus of much attention as a consequence of improved biotechnological capabilities, an improved understanding of lantibiotic biosynthesis and mode of action, and their high specific activity against multi-drug resistant bacteria. This review concerns the fundamental analyses that have revealed the importance of individual amino acids in these peptides and has permitted the implementation of rational mutagenesis strategies ('intelligenetics') to alter individual residues with a view to ultimately widening the active pH range, improve stability, and enhance binding to cell wall targets with the ultimate aim of optimising their antimicrobial activity. It is hoped that as a consequence of this improved knowledge the most suitable application of individual lantibiotics will become apparent. It should also prove possible, in the near future, to generate tailor-made lantibiotics and utilise biosynthetic enzymes to incorporate modified amino acids into non-lantibiotic peptides. In the shorter term, the extensive characterisation of lantibiotics will be instrumental in reassuring drug industry regulators of their safety and facilitate the widespread application of these novel antimicrobial agents in medicine.     

具有双活性序列的新型抗菌肽的设计及性质

设计合成了具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性、 细胞毒性及溶血性. 结果表明, 线性肽和环状肽的杀菌活性高于短链肽. 利用计算模拟的方法计算了多肽与细菌细胞膜中一种重要的成分磷脂酰甘油(DMPG)的结合能. 结果表明, 多肽-DMPG的结合能与多肽的杀菌活性具有较高的相关性, 线性和环状多肽与DMPG的结合能大于短链肽. 线性和环状多肽均含有2个活性序列, 可提供多个荷正电氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 采用模拟生物膜对其中几条多肽的作用机理进行了初步研究. 结果表明, 该类多肽有可能使正常哺乳动物细胞的细胞膜产生孔洞; 而对于细菌细胞膜, 多肽并未在膜上产生明显孔洞, 而是引起了细菌细胞膜的聚集.     

Solution Structure and Model Membrane Interactions of P‐113, a Clinically Active Antimicrobial Peptide Derived from Human Saliva

P‐113, AKRHHGYKRKFH‐NH₂, was derived from human saliva and found to possess clinical activity against fungus infections in HIV patients with oral candidiasis. We have determined the solution structure of P‐113 bound to membrane‐mimetic SDS micelles by two‐dimensional NMR methods. The SDS micelle‐bound structure of P‐113 adopts an α‐helical segment and the positively charged residues are clustered together to form a hydrophilic patch. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy and microcalorimetry, were used to show that P‐113 interacted with negatively charged phospholipid vesicles and induced dye release from these vesicles. However, its dye leakage efficiency is much less than the results of previously reported antimicrobial peptides. These results suggest that the antimicrobial activity of P‐113, unlike other antimicrobial peptides, may act not only through binding to and destabilization of the microbial membrane but also through a specific protein receptor on the microbial cell surface.     

De novo design, synthesis, and characterization of antimicrobial beta-peptides

beta-Peptides are a class of polyamides that have been demonstrated to adopt a variety of helical conformations. Recently, a series of amphiphilic L(+2) helical beta-peptides were designed, which were intended to mimic the overall physicochemical properties of a class of membrane-active antimicrobial peptides, including magainin and cecropin. Although these peptides showed potent antimicrobial activity, they also showed significant activity against human erythrocytes. Operating under the assumption that their lack of specificity arose from excessive hydrophobicity, two additional beta-peptides H-(beta(3)-HAla-beta(3)-HLys-beta(3)-HVal)(n)-NH(2) (n = 4, 5) were designed and synthesized. Both have high antimicrobial activities, but very low hemolytic potencies. The peptides bind in an L(+2) conformation to phospholipid vesicles, inducing leakage of entrapped small molecules. The peptides have a low affinity for membranes consisting of neutral phosphatidylcholine lipids, but bind avidly to vesicles containing 10 mol % of acidic phosphatidylserine lipids. Differences in vesicle leakage kinetics for the two peptides suggest that chain length could affect their mechanisms of disrupting cell membranes. Thus, insights gained from the study of variants of natural alpha-peptides have provided a useful guide for the design of nonnatural antimicrobial beta-peptides.     

Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides

We have designed beta-amino acid oligomers that are helical, cationic, and amphiphilic with the intention of mimicking the biological activity of amphiphilic, cationic alpha-helical antimicrobial peptides found in nature (e.g., magainins). We have previously identified a 17-residue beta-peptide (called beta-17) with antibiotic activity similar to that of a magainin derivative against four bacterial species, including two clinical isolates that are resistant to common antibiotics. This beta-peptide displays very low hemolytic activity against human red blood cells, which indicates selectivity for bacterial cells over mammalian cells. Here we examine some of the factors important for activity in this class of beta-peptides. An amphiphilic helix is necessary, because a nonamphiphilic isomer proved to be inactive. The ratio of cationic to hydrophobic residues is also important. Active beta-peptides induce the leakage of beta-galactosidase from treated Bacillus subtilis cells, as do alpha-helical antibiotic peptides, and this similarity suggests that the beta-peptide mode of action involves disruption of microbial membranes. This class of beta-peptides is not degraded by proteases, which bodes well for biological applications.     

Cathelicidins--nature's attempt at combinatorial chemistry

Cathelicidins are a family of diverse antimicrobial peptides found in granules of mammalian neutrophils. Cathelicidins are active against a broad range of microbes in different environments. Aside from their antimicrobial activity, cathelicidins possess other biological properties including cytotoxic activity towards mammalian cells. Several studies have shown that the amino acid sequence of cathelicidins can be modified to temper undesired properties, such as hemolytic and cytotoxic activity, and at the same time maintain antimicrobial activity. These properties make cathelicidins ideal templates in combinatorial chemistry for designing de novo antimicrobial peptides for therapeutic use. However, one of the major challenges will be to screen these peptides in experimentally relevant models that reflect the environments in which the peptides should be therapeutically active.