Current Protein and Peptide Science - Volume 11, Issue 3, 2010

The central idea of this issue is to describe and discuss the potentialities of antimicrobial molecules isolated from native plant species of Brazil known to be employed by Amerindian populations as powerful antimicrobial drugs used in the traditional popular medicine, and from plants with great commercial and social interest such as sugarcane and legumes, respectively used for sugar as well as biofuels production and basic daily people feed. The enormous biodiversity of these plants represents a very powerful target to new drugs searching. The objective is to describe the progresses on seeking for new molecules with potential antimicrobial activity and new antimicrobial peptides able to fight against pathogen infections. The different topics of this issue describe the genomic and post-genomic strategies used to isolate new genes encoding for antimicrobial peptides, the in silico search for new antimicrobial molecules, the strategies for protein data mining in public databases and the suitable technologies for plant-derived antimicrobial peptides synthesis. The extraordinary biodiversity of native plants in Brazil, known to be used as alternative medicine approach by native Amerindian populations, is described in the Ethnobotanic topic, while epidemiological and pharmacological studies evidence several plant species as potential candidates for the development of new alternative drugs which are supposed to protect mucosal surfaces from microbes entrance and establishment. The approach described for identification of new interesting antimicrobial molecules relies on searching in silico the genes putatively coding for potential antimicrobial molecules already reported in public or in some private (such as SuperSAGE libraries from tolerant cultivated genotypes after pathogen infection) plant databases. The availability of such data shall substantially enhance understanding of genes and enzymes associated with natural products biosynthesis. The isolation of these molecules from native plants (where they have never been isolated from), the synthesis of these molecules and finally the in vitro testing for the potential antimicrobial activity will provide the researchers with better tools to fight against pathogens. Moreover, attention is focused on plants harboring social and economical relevance such as the sugarcane and members of the legume family. In fact sugarcane represents a central point in Brazilian northeastern economy since it is used to produce mainly the alternative biofuel ethanol and sugar, while the legumes are important part of the widely used popular food. The isolation of new antimicrobial peptides from these plants will allow the improvement of strategies for struggle against parasites and could reduce the negative effects of pesticides on the agricultural workers and the environment. The great efforts of the scientific community for the discovery of new drugs or nutraceuticals are nowadays directioned to the search of “natural” molecules, sometimes already known by ancient or traditional popular medicine or agriculture as efficient antimicrobials. The joint efforts including ethnobotanic, molecular biology and evolutionary genetics, biochemistry and cellular biology as well as epidemiology and bioinformatics are turning available the identification of a growing number of antimicrobial molecules with interesting potential applications; in this issue we discuss the different research strategies aiming the identification of antimicrobial molecules derived from native and crop Brazilian plant species and the potentiality of the results obtained so far.

Mechanisms related to biotic interactions, such as pathogen attack, herbivory and symbiosis are important challenges to higher plants and have been widely studied especially for breeding purposes. The present review focuses on a special category of defense molecules, the plant antimicrobial peptides, providing an overview of their main molecular features and structures.

Despite of the high biological diversity and traditional use of medicinal plants in Brazil, no comprehensive ethnobotanic review of plants with potential antimicrobial effects is available. In the present work own field information is aggregated with a literature review, identifying 433 Brazilian plant species potentially useful for identification of antimicrobial peptides. They included mainly woody species, distributed on 100 plant families (93 angiosperms and 7 pteridophytes) and 266 genera, covering all Brazilian regions and ecosystems. Main plant parts and indications for their use are presented and discussed, revealing the high potential that these plants present for the future planning strategies regarding the future development of antimicrobial drugs.

The extensive amount of nucleotide sequences from diverse plant species in Data Banks enables the use of computational approaches to discovery still unidentified genes and to infer about their function, structure and role in some biological processes. Of special interest are the antimicrobial peptides (AMP), whose functionalities have a very important role in defense against microbial infection in multicellular eukaryotes, being considered less susceptible to bacterial resistance than traditional antibiotics, with potential to develop a new class of therapeutic agents. Recent computational developments have provided various algorithms and resources to profit from the overwhelming information in data banks for biomining such peptides. This review focuses on the computational and bioinformatic approaches so far used for the identification of antimicrobial peptides in plant systems, highlighting alternative means of mining the entire plant peptide space that has recently become available.

As eukaryotes, plants include in innate defense antimicrobial peptides (AMP), usually small cysteine or glycine- rich peptides effective against a wide range of pathogens. The main classes of AMPs are represented by α/β- defensins, lipid-transfer proteins, thionins, cyclotides, snakins and hevein-like, according to amino acid sequence homology. In spite of increasing number of described AMPs from plants, last decade advances in methodologies for gene expression and the huge amounts of genomic, proteomic and other “-omics” data lead to new prospection strategies of novel potential candidates. Organised user-friendly databases are available to be searched and enlarged with newly discovered plant-derived AMPs. Bioinformatics has allowed the application of in silico-associated molecular tools aiming to screen and identify genes coding for these peptides, starting from genome, transcriptomes, proteome or metabolome from various cultivated or wild plants. As expected, crop plants have been the main target for AMP research and application, also because the higher availability of molecular data. However, wild plant species biodiversity and results for AMP search have increased the importance of characterization in native plants. Enormous plant diversity in Brazilian ecosystems summed to croplands provides potential targets to identify novel candidates for plant AMP. Despite these opportunities, bioinformatics tools are restricted to species whose “-omics” are available, otherwise only heterology-based analyses are feasible, as it has been the case of most Brazilian plant AMP prospection research groups. Still rare, but promising results indicate that this research field on Brazilian crop/native species presents a growing trend of application in agriculture, medicine and industry.

Antimicrobial peptides exert an important role in plant defence and their structure/activity relationship against pathogens is widely described. Although the most striking feature of these antimicrobial peptides is their molecular diversity, they share some common features, such as a relatively low molecular weight, and the presence of a variable number of cysteines residues that contribute to stabilize conserved scaffolds through disulphide bond formation, and can be assigned to different structural classes. Peptides from different classes in some cases act synergistically against pathogens when produced by the same tissue, and contribute to extending defence to a wider range of microbes. In this review we briefly describe the structure of some of the main plant antimicrobial peptide classes: thionins, defensins, lipid transfer proteins, cyclotides and snakins, and how they are reported to contribute to the plant protection. In many cases these antimicrobial peptides show a wider activity spectrum than that suggested by their name, exerting an action also against predatory insects and revealing useful antiviral activities. This extends their interest from defense of important food crops also to the design of novel anti-infective compounds for both pharmaceutical and agricultural applications.

Defensin, thionin and lipid transfer protein (LTP) gene families, which antimicrobial activity has an attractive use in protein engineering and transgenic production of agronomically important plants, have been here functionally reviewed. Also, a transcriptional overview of plant SuperSAGE libraries and analysis of 26 bp tags possibly annotated for those families are presented. Tags differentially expressed (p < 0.05) or constitutively transcribed were identified from leaves or roots from SuperSAGE libraries from important Brazilian plant crops [cowpea (Vigna unguiculata (L.) Walp.), soybean (Glycine max (L.) Merr.) and modern sugarcane hybrids (Saccharum spp.)] submitted to abiotic [salt (100 mM NaCl) or drought] or biotic stresses [fungus inoculation (Phakopsora pachyrhizi; Asiatic Soyben Rust phytopathogen)]. The diverse transcriptional patterns observed, probably related to the variable range of targets and functions involved, could be the first step to unravel the antimicrobial peptide world and the plant stress response relationship. Moreover, SuperSAGE opens the opportunity to find some SNPs or even rare transcripts that could be important on plant stress resistance mechanisms. Putative defensin or LTPs revealed by SuperSAGE following a specific plant treatment or physiological condition could be useful for future use in genetic improvement of plants.

To defend themselves from attack by pathogens, plants can rely only on their innate defense systems. Defensins are antimicrobial peptides that contribute to plant immunity by displaying a direct cidal activity against various pathogens, some of which are responsible for plant diseases. These determine a significant decrease in the quality and safety of agricultural products, especially among food crops, and cause significant economic losses. There is consequently an increasing interest for antimicrobial compounds such as the defensins, which might be applied in different ways to protect important food or bio-fuel crops. In this review we analyze the techniques that have been reported in the literature for the production of isolated plant defensins of adequate quality and sufficient quantity for potential use in plant protection. For research purposes, defensins have been heterologously expressed in diverse hosts, such as bacteria, yeasts, fungi and plants. Chemical synthesis is instead not commonly used for their production, due to structural characteristics that make it difficult to obtain the correct protein folding. To consider the possibility of producing plant defensins in a large scale, cost-effective methods guaranteeing high quality product are required. Future studies may thus focus on the development of more stable compounds, as well as decreasing production costs by improving preparative syntheses or biotechnological procedures such as using transgenic crops as plant factories.

All living organisms, ranging from microorganisms to plants and mammals, have evolved mechanisms to actively defend themselves against pathogen attack. A wide range of biological activities have been attributed to plant antimicrobial peptides (AMPs) including growth inhibitory effects on a broad range of fungi, Gram-positive and Gram-negative bacteria, viruses, neoplastic cells and parasitic protozoa. Classes of AMPs, their mechanisms of action, biological activity, and cytotoxicity towards host cells are discussed. A particular focus regards AMP candidates with potential for use in defense against biological warfare agents. This field is young, but provides additional stimulus to consideration of these molecules as a new class of therapeutic agents and promises to revolutionize treatment of many infectious diseases.

EST (Expressed Sequence Tags) databases are increasing in number and size, especially regarding cultivated plants. Sugarcane is one of the most important tropical and subtropical crops, presenting a complex polyploid genome of hybrid origin, bearing a challenge for the understanding of genetic processes in higher plants. In the present work a general search was carried out on the largest Sugarcane EST Database (SUCEST) that includes 237,954 ESTs aiming to identify defensin antimicrobial peptides - a class of small, basic, cysteine-rich peptides distributed throughout the kingdoms. Using a computational approach 17 new defensin isoforms could be identified. Main steps for the search, characterization and evaluation of the defensin expression profile are presented. Prevalent expression tissues were leaf roll, lateral bark, root apex, base of inflorescence, developing seed, and calli. Bioinformatics and phylogenetic analysis of the primary structure of the sugarcane defensin candidates as well as the 3D structures obtained by comparative modeling support their role as antimicrobials.