Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

Joker: An algorithm to insert patterns into sequences for designing antimicrobial peptides

Innovative alternatives to control bacterial infections are need due to bacterial resistance rise. Antimicrobial peptides (AMPs) have been considered as the new generation of antimicrobial agents. Based on the fact that AMPs are sequence-dependent, a linguistic model for designing AMPs was previously developed, considering AMPs as a formal language with a grammar (patterns or motifs) and a vocabulary (amino acids). Albeit promising, that model has been poorly exploited mainly because thousands of sequences need to be generated, and the outcome has high similarity to already known AMPs. Here we present Joker, an innovative algorithm that improves the application of the linguistic model for rational design of antimicrobial peptides. We modelled the AMPs as a card game, where Joker combines the cards in the hand (patterns) with the cards in the table (sequence templates), generating a few variants. Our algorithm is capable of improving existing AMPs or even creating new AMPs from inactive peptides. A standalone version of Joker is available for download at <http://github.com/williamfp7/Joker> and requires a Linux 32-bit machine.     

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice

The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.     

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.     

Fast and potent bactericidal membrane lytic activity of PaDBS1R1, a novel cationic antimicrobial peptide

Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5?μM) and Gram-positive (MIC of 3?μM) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25?μM) and methicillin-resistant Staphylococcus aureus (MIC of 12.5?μM), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy.     

Discovery of potent antimicrobial peptide analogs of Ixosin-B

Antimicrobial peptides (AMPs) represent the first defense line against infection when organisms are infected by pathogens. These peptides are generally good targets for the development of antimicrobial agents. Peptide amide analogs of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, were designed, synthesized and examined for antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Within the peptides synthesized, we discovered an 11-mer peptide, KRLRRVWRRWR-amide, which exhibited potent antimicrobial activity while very little hemolytic activity in human erythrocytes was observed even at high dose level (100 μM). With further modifications, this peptide could be developed into a potent antimicrobial agent in the future.     

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog,Rana nigrovittata

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents.     

Two novel families of antimicrobial peptides from skin secretions of the Chinese torrent frog, Amolops jingdongensis

The characterization of new natural antimicrobial peptides (AMPs) can help to solve the serious problem of bacterial resistance to currently used antibiotics. In the current study, we analyzed two families of AMPs from the Chinese torrent frog Amolops jingdongensis with a range of bioactivities. The first family of peptides, named jindongenin-1a, is 24 amino acids in length; a BLAST search of jindongenin-1a revealed no sequence similarity with other AMPs. The second family consists of two peptides containing 29 amino acid residues each. These peptides have high sequence similarity with the AMPs of palustrin-2 and are therefore designated palustrin-2AJ1 and palustrin-2AJ2. The cDNA sequences encoding these AMPs have been cloned and the deduced protein sequence of each AMP has been determined by protein sequencing. Sequence and structural analysis showed that each precursor is composed of a putative signal peptide, an N-terminal spacer, a processing site and a disulfide-bridged heptapeptide segment at the C-terminus. We synthesized jindongenin-1a and palustrin-AJ1 to test their antimicrobial, hemolytic, antioxidative and cytotoxic activities. These two peptides showed broad-spectrum antimicrobial activity to standard and clinically isolated strains of bacteria. In addition, they exhibited weak hemolytic activity to human and rabbit erythrocytes under our experimental conditions. Moreover, these peptides also displayed cytotoxic activity against the K562 and HT29 mammalian cell lines and low anti-oxidant activity. These findings provide helpful insight that will be useful in the design of anti-infective peptide agents.     

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks.     

Differential interactions of the antimicrobial peptide,RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes

BackgroundAntimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus.MethodsA physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations.ResultsRQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces.ConclusionsRQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application.General significanceThese results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.     

Membrane-active and DNA binding related double-action antimycobacterial mechanism of antimicrobial peptide W3R6 and its synthetic analogs

The emergence of multidrug- or extremely drug-resistant M. tuberculosis strains has made very few drugs available for current tuberculosis treatment. Antimicrobial peptides can be employed as a promising alternative strategy for TB treatment. Here, we designed and synthesized a series of peptide sequences based on the structure-activity relationships of natural sequences of antimicrobial peptides. The peptide W3R6 and its analogs were screened and found to have potent antimycobacterial activity against M. smegmatis, and no hemolytic activity against human erythrocytes. The evidence from the mechanism of action study indicated that W3R6 and its analogs can interact with the mycobacterial membrane in a lytic manner and form pores on the outer membrane of M. smegmatis. Significant colocalization of D-W3R6 with mycobacterial DNA was observed by confocal laser scanning microscopy and DNA retardation assays, which suggested that the antimycobacterial mechanism of action of the peptide was associated with the unprotected genomic DNA of M. smegmatis. In general, W3R6 and its analogs act on not only the mycobacterial membrane but also the genomic DNA in the cytoplasm, which makes it difficult for mycobacteria to generate resistance due to the peptides having two targets. In addition, the peptides can effectively eliminate M. smegmatis cells from infected macrophages. Our findings indicated that the antimicrobial peptide W3R6 could be a novel lead compound to overcome the threat from drug-resistant M. tuberculosis strains in the development of potent AMPs for TB therapeutic applications.     

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa).The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition.We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.     

景东湍蛙抗菌肽jindongenin-1d的分析和活性测定

新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。     

Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negative bacteria by fusion with LPS-targeting peptide

Antimicrobial peptides (AMPs) provide a potential source of new antimicrobial therapeutics for the treatment of multidrug-resistant pathogens. To develop Gram-negative selective AMPs that can inhibit the effects of lipopolysaccharide (LPS)-induced sepsis, we added various rationally designed LPS-targeting peptides [amino acids 28–34 of lactoferrin (Lf28–34), amino acids 84–99 of bactericidal/permeability increasing protein (BPI84–99), and de novo peptide (Syn)] to the potent AMP, GNU7 (RLLRPLLQLLKQKLR). Compared to our original starting peptide GNU7, hybrid peptides had an 8- to 32-fold improvement in antimicrobial activity against Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium. Among them, Syn-GNU7 showed the strongest LPS-binding and -neutralizing activities, thus allowing it to selectively eliminate Gram-negative bacteria from within mixed cultures. Our results suggest that LPS-targeting peptides would be useful to increase the antimicrobial activity and selectivity of other AMPs against Gram-negative bacteria.     

Anticancer activities of an antimicrobial peptide derivative of Ixosin-B amide

In nature, antimicrobial peptides (AMPs) represent the first line of defense against infection by pathogens; thus, they are generally good candidates for the development of antimicrobial agents. Recently, we reported two potent antimicrobial peptides, KWLRRVWRWWR-amide (MAP-04-03) and KRLRRVWRRWR-amide (MAP-04-04), which were derived from a fragment of Ixosin-B-amide (KSDVRRWRSRY). Since some cationic AMPs exhibited cytotoxic activity against cancer cells, in the current study, we further investigated the anticancer activity of these potent antimicrobial peptides by antiproliferative assays and wound-healing assays, and the effect of peptide on the cytoskeleton alteration and cell morphology were analyzed by confocal microscopy. Results indicated that MAP-04-03 not only exhibited inhibitory effects on the proliferation (IC₅₀ = 61.5 μM) and on the cell migration of MCF-7 breast cancer cells (at a concentration of 5 μM), but also affected the cytoskeleton at the concentration of 25 μM. These results demonstrated that MAP-04-03 can serve as a lead peptide analog for developing potent anticancer agents.     

Connecting peptide physicochemical and antimicrobial properties by a rational prediction model

The increasing rate in antibiotic-resistant bacterial strains has become an imperative health issue. Thus, pharmaceutical industries have focussed their efforts to find new potent, non-toxic compounds to treat bacterial infections. Antimicrobial peptides (AMPs) are promising candidates in the fight against antibiotic-resistant pathogens due to their low toxicity, broad range of activity and unspecific mechanism of action. In this context, bioinformatics' strategies can inspire the design of new peptide leads with enhanced activity. Here, we describe an artificial neural network approach, based on the AMP's physicochemical characteristics, that is able not only to identify active peptides but also to assess its antimicrobial potency. The physicochemical properties considered are directly derived from the peptide sequence and comprise a complete set of parameters that accurately describe AMPs. Most interesting, the results obtained dovetail with a model for the AMP's mechanism of action that takes into account new concepts such as peptide aggregation. Moreover, this classification system displays high accuracy and is well correlated with the experimentally reported data. All together, these results suggest that the physicochemical properties of AMPs determine its action. In addition, we conclude that sequence derived parameters are enough to characterize antimicrobial peptides.     

Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli

Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H₂O₂. Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.     

Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides

The presence of an effective immune response in the hemocoel of ticks is crucial for survival, as it prevents the invasion of pathogens throughout the animal's body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a subtraction hybridization cDNA library was constructed from the salivary glands of the unfed and fed female tick Rhipicephalus haemaphysaloides, and a novel cysteine-rich AMP designated Rhamp (R. haemaphysaloides antimicrobial peptide) was isolated and identified. The Rhamp was encoded by a gene with an open reading frame of 303 bp which encoded a mature peptide with 8 kDa molecular weight. No identity was found by BLAST search to any database entries. The sequence encoding the Rhamp was subcloned into the pGEX-4T vector and expressed in Escherichia coli. The recombinant protein of Rhamp showed chymotrypsin and elastase-inhibitory activity and markedly inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli. Moreover, the recombinant protein also exerted low hemolytic activity. These results indicate the Rhamp is a novel antimicrobial peptide with proteinase activity from the tick R. haemaphysaloides.