Knowledge-based computational methods for identifying or designing novel,non-homologous antimicrobial peptides

We describe computational approaches for identifying promising lead candidates for the development of peptide antibiotics, in the context of quantitative structure–activity relationships (QSAR) studies for this type of molecule. A first approach deals with predicting the selectivity properties of generated antimicrobial peptide sequences in terms of measured therapeutic indices (TI) for known antimicrobial peptides (AMPs). Based on a training set of anuran AMPs, the concept of sequence moments was used to construct algorithms that could predict TIs for a second test set of natural AMPs and could also predict the effect of point mutations on TI values. This approach was then used to design peptide antibiotics (adepantins) not homologous to known natural or synthetic AMPs. In a second approach, many novel putative AMPs were identified from DNA sequences in EST databases, using the observation that, as a rule, specific subclasses of highly conserved signal peptides are associated exclusively with AMPs. Both anuran and teleost sequences were used to elucidate this observation and its implications. The predicted therapeutic indices of identified sequences could then be used to identify new types of selective putative AMPs for future experimental verification.     

Screening and cloning of antimicrobial DNA sequences using a vital staining method

A novel method for cloning of genes coding for cytotoxic molecules based on a cell viability assay is described. The working hypothesis is that expression of DNA sequences coding for cytotoxic molecules in bacterial cells will lead to cell death or impairment, and the isolation of the impaired or dead cells could lead to identification of DNA sequences responsible for debilitating the host cells. We verified this concept by isolating the well known antimicrobial Puroindoline b gene in Escherichia coli cells. We further demonstrated the feasibility to use this approach for isolating DNA encoding for antimicrobials from cDNA expression libraries. Sequence analysis and bioassay indicated that the isolated clones encoded previously characterized antimicrobial proteins (AMPs), proteins not previously characterized as AMPs, as well as novel antimicrobial peptides. In addition, clones harboring ribosomal protein encoding cDNA were also identified. Therefore, this method could also be used to identify host genes important in maintaining bacterial cell viability.     

蛋白质组学中新蛋白质鉴定的研究方法和策略

当前,基于生物质谱进行蛋白质鉴定的技术已经成为蛋白质组学研究的支撑技术之一.产生的数据主要使用数据库搜索的方法进行处理,这种方法的一大缺陷是不能鉴定数据库中未包含的蛋白质,因此如何充分利用质谱数据对蛋白质组研究的意义很大,而新蛋白质鉴定更是其中一个重要的内容.新蛋白质鉴定是蛋白质鉴定的一个方面,新蛋白质的定义按照序列和功能的已知程度分为3个层次;以蛋白质鉴定的方法为基础,目前新蛋白质鉴定的方法可分为denovo测序和相似序列搜索结合的方法以及搜索EST、基因组等核酸数据库的方法2大类;两者各有利弊.存在各自的问题和相应处理的策略.不同的研究者可以根据具体目的应用和发展不同的鉴定方法,同时新蛋白质的鉴定也将随着蛋白质组学研究的发展而更加完善.     

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae)

As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.     

Antimicrobial Peptides: Successes, Challenges and Unanswered Questions

Multidrug antibiotic resistance is an increasingly serious public health problem worldwide. Thus, there is a significant and urgent need for the development of new classes of antibiotics that do not induce resistance. To develop such antimicrobial compounds, we must look toward agents with novel mechanisms of action. Membrane-permeabilizing antimicrobial peptides (AMPs) are good candidates because they act without high specificity toward a protein target, which reduces the likelihood of induced resistance. Understanding the mechanism of membrane permeabilization is crucial for the development of AMPs into useful antimicrobial agents. Various models, some phenomenological and others more quantitative or semimolecular, have been proposed to explain the action of AMPs. While these models explain many aspects of AMP action, none of the models captures all of the experimental observations, and significant questions remain unanswered. Here, we discuss the state of the field and pose some questions that, if answered, could speed the discovery of clinically useful peptide antibiotics.     

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.     

AMPer: a database and an automated discovery tool for antimicrobial peptides

MOTIVATION: Increasing antibiotics resistance in human pathogens represents a pressing public health issue worldwide for which novel antibiotic therapies based on antimicrobial peptides (AMPs) may offer one possible solution. In the current study, we utilized publicly available data on AMPs to construct hidden Markov models (HMMs) that enable recognition of individual classes of antimicrobials peptides (such as defensins, cathelicidins, cecropins, etc.) with up to 99% accuracy and can be used for discovering novel AMP candidates. RESULTS: HMM models for both mature peptides and propeptides were constructed. A total of 146 models for mature peptides and 40 for propeptides have been developed for individual AMP classes. These were created by clustering and analyzing AMP sequences available in the public sources and by consequent iterative scanning of the Swiss-Prot database for previously unknown gene-coded AMPs. As a result, an additional 229 additional AMPs have been identified from Swiss-Prot, and all but 34 could be associated with known antimicrobial activities according to the literature. The final set of 1045 mature peptides and 253 propeptides have been organized into the open-source AMPer database. AVAILABILITY: The developed HMM-based tools and AMP sequences can be accessed through the AMPer resource at http://www.cnbi2.com/cgi-bin/amp.pl     

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken

Antimicrobial peptides (AMPs) are essential components of innate immunity in a range of species from Drosophila to humans and are generally thought to act by disrupting the membrane integrity of microbes. In order to discover novel AMPs in the chicken, we have implemented a bioinformatic approach that involves the clustering of more than 420,000 chicken expressed sequence tags (ESTs). Similarity searching of proteins—predicted to be encoded by these EST clusters—for homology to known AMPs has resulted in the in silico identification of full-length sequences for seven novel gallinacins (Gal-4 to Gal-10), a novel cathelicidin and a novel liver-expressed antimicrobial peptide 2 (LEAP-2) in the chicken. Differential gene expression of these novel genes has been demonstrated across a panel of chicken tissues. An evolutionary analysis of the gallinacin family has detected sites—primarily in the mature AMP—that are under positive selection in these molecules. The functional implications of these results are discussed.     

Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila

To improve the utility of increasingly large numbers of available unannotated and initially poorly annotated genomic sequences for proteome analysis, we demonstrate that effective protein identification can be made on a large and unannotated genome. The strategy developed is to translate the unannotated genome sequence into amino acid sequence encoding putative proteins in all six reading frames, to identify peptides by tandem mass spectrometry (MS/MS), to localize them on the genome sequence, and to preliminarily annotate the protein via a similarity search by BLAST. These tasks have been optimized and automated. Optimization to obtain multiple peptide matches in effect extends the searchable region and results in more robust protein identification. The viability of this strategy is demonstrated with the identification of 223 cilia proteins in the unicellular eukaryotic model organism Tetrahymena thermophila, whose initial genomic sequence draft was released in November 2003. To the best of our knowledge, this is the first demonstration of large-scale protein identification based on such a large, unannotated genome. Of the 223 cilia proteins, 84 have no similarity to proteins in NCBI's nonredundant (nr) database. This methodology allows identifying the locations of the genes encoding these novel proteins, which is a necessary first step to downstream functional genomic experimentation.     

Modulation of free energy landscapes as a strategy for the design of antimicrobial peptides

Computational design of antimicrobial peptides (AMPs) is a promising area of research for developing novel agents against drug-resistant bacteria. AMPs are present naturally in many organisms, from bacteria to humans, a time-tested mechanism that makes them attractive as effective antibiotics. Depending on the environment, AMPs can exhibit α-helical or β-sheet conformations, a mix of both, or lack secondary structure; they can be linear or cyclic. Prediction of their structures is challenging but critical for rational design. Promising AMP leads can be developed using essentially two approaches: traditional modeling of the physicochemical mechanisms that determine peptide behavior in aqueous and membrane environments and knowledge-based, e.g., machine learning (ML) techniques, that exploit ever-growing AMP databases. Here, we explore the conformational landscapes of two recently ML-designed AMPs, characterize the dependence of these landscapes on the medium conditions, and identify features in peptide and membrane landscapes that mediate protein-membrane association. For both peptides, we observe greater conformational diversity in an aqueous solvent than in a less polar solvent, and one peptide is seen to alter its conformation more dramatically than the other upon the change of solvent. Our results support the view that structural rearrangement in response to environmental changes is central to the mechanism of membrane-structure disruption by linear peptides. We expect that the design of AMPs by ML will benefit from the incorporation of peptide conformational substates as quantified here with molecular simulations.

     

Antimicrobial peptides (AMPs): A promising class of antimicrobial compounds

Antimicrobial peptides (AMPs) are compounds, which have inhibitory activity against microorganisms. In the last decades, AMPs have become powerful alternative agents that have met the need for novel anti-infectives to overcome increasing antibiotic resistance problems. Moreover, recent epidemics and pandemics are increasing the popularity of AMPs, due to the urgent necessity for effective antimicrobial agents in combating the new emergence of microbial diseases. AMPs inhibit a wide range of microorganisms through diverse and special mechanisms by targeting mainly cell membranes or specific intracellular components. In addition to extraction from natural sources, AMPs are produced in various hosts using recombinant methods. More recently, the synthetic analogues of AMPs, designed with some modifications, are predicted to overcome the limitations of stability, toxicity and activity associated with natural AMPs. AMPs have potential applications as antimicrobial agents in food, agriculture, environment, animal husbandry and pharmaceutical industries. In this review, we have provided an overview of the structure, classification and mechanism of action of AMPs, as well as discussed opportunities for their current and potential applications.     

Cell-penetrating peptides and antimicrobial peptides: how different are they?

Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells; however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented.     

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.     

Sense the moment: A highly sensitive antimicrobial activity predictor based on hydrophobic moment

BackgroundComputer-aided identification and design tools are indispensable for developing antimicrobial agents for controlling antibiotic-resistant bacteria. Antimicrobial peptides (AMPs) have aroused intense interest, since they have a broad spectrum of activity, and therefore, several systems for predicting antimicrobial peptides have been developed, using scalar physicochemical properties; however, regardless of the machine learning algorithm, these systems often fail in discriminating AMPs from their shuffled versions, leading to the need for new training methods to overcome this bias. Aiming to solve this bias, here we present “Sense the Moment”, a prediction system capable of discriminating AMPs and shuffled versions.MethodsThe system was trained using 776 entries: 388 from known AMPs and another 388 based on shuffled versions of known AMPs. Each entry contained the geometric average of three hydrophobic moments measured with different scales.ResultsThe model showed good accuracy (>80%) and excellent sensitivity (>90%) for AMP prediction, exceeding deep-learning-based methods.ConclusionOur results demonstrate the system's applicability, aiding in identifying and discarding non-AMPs, since the number of false negatives is lower than false positives.General significanceThe application of this model in virtual screening protocols for identifying and/or creating antimicrobial agents could aid in the identification of potential drugs to control pathogenic microorganisms and in solving the antibiotic resistance crisis.AvailabilityThe system was implemented as a web application, available at <http://portoreports.com/stm/>.     

Applications of antimicrobial peptides from fish and perspectives for the future

Fish are a major component of the aquatic fauna. Like other organisms, fish secrete different kinds of antimicrobial peptides (AMPs), which are positively charged short amino-acid-chain molecules involved in host defense mechanisms. Environmental hazards and the greenhouse effect have led to increased evolution of drug- and vaccine-resistant pathogenic strains, and it is necessary to find new drugs with structural uniqueness to fight them. Aquatic sources contain thousands of fish species, and each secretes AMPs with structural differences which can be used by the pharmaceutical industry in its search for novel drugs to treat drug-resistant pathogens. Not only limited to antimicrobial functions, AMPs possess other desirable characteristics which may be exploited in the near future. In this review, we list fish AMPs available from published reports, and discuss application-oriented functions of these AMPs. Notably, the possibilities of using fish AMPs as antimicrobial agents, vaccine adjuvants, inactivated vaccines, and antitumor agents are discussed in this review.     

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.     

Bacteriocins: perspective for the development of novel anticancer drugs

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

     

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.     

The expanding scope of antimicrobial peptide structures and their modes of action

Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure-activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents.