PvD1 defensin,a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis

Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD₁ from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD₁ could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD₁ defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD₁ defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.     

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.     

三条新颖短肽筛选及抑菌作用的初报

[目的]为了发现新的农作物病原菌抗菌肽,人工设计并构建了大容量短肽文库,从中筛选并合成96条短肽用于鉴定其对农作物病原菌的抑菌活性.[方法]采用琼脂扩散法,对靶标菌一棉花枯萎病菌(Fusarium f.sp.vasinfecum)、棉花红腐病菌(Fusarium moniliforme)、小麦根腐病菌(Bipolaris sorokiniana)和马铃薯早疫病菌(Alternaria solani)进行抑菌初筛,并测定了有抗菌作用短肽的最小抑菌浓度和抑菌持久性.[结果]得到了A6、D4和F10对上述四种病原真菌抑菌效果较强,抑菌时间较长的抗菌肽,通过与抗菌肽数据库氨基酸序列对比,未见这3条抗菌肽的同源序列.[结论]研制的3条短肽属于新颖抗菌肽,为防治农作物真菌病害提供了新的基因资源.     

Increased potency of a novel d-β-naphthylalanine-substituted antimicrobial peptide against fluconazole-resistant fungal pathogens

The antifungal activities of the known antimicrobial peptide, P-113, as well as a new type of Trp-rich peptide, Ac-KWRRWVRWI-NH₂, Pac-525, and its modified peptide, d -Nal-Pac-525, were determined using the broth microdilution method in three different media. All peptides had similar activities against yeast pathogens in low-salt LYM media. However, only d -Nal-Pac-525 retained its antifungal activity in the media containing high concentrations of salt. Hence, d -Nal-Pac-525 has the potential of becoming a promising antifungal agent, especially for fungal pathogens with intrinsic resistance to fluconazole.     

De-Novo Design of Antimicrobial Peptides for Plant Protection

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents.     

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides

Plant defensins are small basic peptides that are inhibitory against a range of plant and human pathogens. Their in vitro antimicrobial activity and structural similarity with human and insect defensins indicated an important role for plant defensins in the innate immune system of plants. Regarding their mode of antimicrobial action, most plant defensins interact with a specific microbial surface receptor, resulting in microbial cell death via e.g. induction of apoptosis. However, accumulating evidence suggests additional in vivo functions of these plant defensins, and by extension of the more recently discovered defensin-like peptides, in general plant development. In this review we will discuss both, the functional roles of defensins in the plant and their modes of antimicrobial action.     

Expression of a polyphemusin variant in transgenic tobacco confers resistance against plant pathogenic bacteria,fungi and a virus

Antimicrobial cationic peptides provide a promising means of engineering plant resistance to a range of plant pathogens, including viruses. PV5 is a synthetic structural variant of polyphemusin, a cationic peptide derived from the horseshoe crab-Limulus polyphemus. PV5 has been shown to be benign toward eukaryotic membranes but with enhanced antimicrobial activity against animal pathogens. In this work, the cytotoxicity of PV5 toward tobacco protoplasts and leaf discs was assessed using TTC (2,3,5-triphenyltetrazolium chloride) and Evans blue colorimetric assays. PV5 showed no measurable cytotoxic effects even at levels as high as 60 μg. As a possible approach to enhancing plant resistance, a gene encoding PV5 was fused to the signal sequence encoding the C-terminus portion of the BiP protein from Pseudotsuga menziesii, under the control of 2 × 35S CaMV promoter. When introduced into Nicotiana tabacum var Xanthi gene integration and expression was confirmed by both Southern and northern analyses. When transgenic plants were subsequently challenged with bacterial and fungal phytopathogens enhanced resistance was observed. Moreover, transgenic plants also displayed antiviral properties against Tobacco Mosaic Virus making PV5 an excellent candidate for conferring unusually broad spectrum resistance to plants and the first anti-plant virus antimicrobial peptide.     

Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides

Plant defensins are small basic peptides that are inhibitory against a range of plant and human pathogens. Their in vitro antimicrobial activity and structural similarity with human and insect defensins indicated an important role for plant defensins in the innate immune system of plants. Regarding their mode of antimicrobial action, most plant defensins interact with a specific microbial surface receptor, resulting in microbial cell death via e.g. induction of apoptosis. However, accumulating evidence suggests additional in vivo functions of these plant defensins, and by extension of the more recently discovered defensin-like peptides, in general plant development. In this review we will discuss both, the functional roles of defensins in the plant and their modes of antimicrobial action.     

Plant integrity: An important factor in plant-pathogen interactions

The effect of plant integrity and of aboveground-belowground defense signaling on plant resistance against pathogens and herbivores is emerging as a subject of scientific research. There is increasing evidence that plant defense responses to pathogen infection differ between whole intact plants and detached leaves. Studies have revealed the importance of aboveground-belowground defense signaling for plant defenses against herbivores, while our studies have uncovered that the roots as well as the plant integrity are important for the resistance of the potato cultivar Sarpo Mira against the hemibiotrophic oomycete pathogen Phytophthora infestans. Furthermore, in the Sarpo Mira–P. infestans interactions, the plant’s meristems, the stalks or both, seem to be associated with the development of the hypersensitive response and both the plant’s roots and shoots contain antimicrobial compounds when the aerial parts of the plants are infected. Here, we present a short overview of the evidence indicating the importance of plant integrity on plant defense responses.     

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

Antibiotic resistance is a major public health problem globally. Particularly concerning amongst drug‐resistant human pathogens is Mycobacterium tuberculosis that causes the deadly infectious tuberculosis (TB) disease. Significant issues associated with current treatment options for drug‐resistant TB and the high rate of mortality from the disease makes the development of novel treatment options against this pathogen an urgent need. Antimicrobial peptides are part of innate immunity in all forms of life and could provide a potential solution against drug‐resistant TB. This review is a critical analysis of antimicrobial peptides that are reported to be active against the M tuberculosis complex exclusively. However, activity on non‐TB strains such as Mycobacterium avium and Mycobacterium intracellulare, whenever available, have been included at appropriate sections for these anti‐TB peptides. Natural and synthetic antimicrobial peptides of diverse sequences, along with their chemical structures, are presented, discussed, and correlated to their observed antimycobacterial activities. Critical analyses of the structure allied to the anti‐mycobacterial activity have allowed us to draw important conclusions and ideas for research and development on these promising molecules to realise their full potential. Even though the review is focussed on peptides, we have briefly summarised the structures and potency of the various small molecule drugs that are available and under development, for TB treatment.     

Novel short antibacterial and antifungal peptides with low cytotoxicity: Efficacy and action mechanisms

Short antimicrobial peptides with nine and eleven residues were developed against several clinically important bacterial and fungal pathogens (specifically Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Fusarium solani). Twelve analogues of previously reported peptides BP76 (KKLFKKILKFL) and Pac-525 (KWRRWVRWI) were designed, synthesized, and tested for their antimicrobial activities. Two of our eleven amino acid peptides, P11-5 (GKLFKKILKIL) and P11-6 (KKLIKKILKIL), have very low MICs of 3.1-12.5 μg ml⁻¹ against all five pathogens. The MICs of these two peptides against S. aureus, C. albicans and F. solani are four to ten times lower than the corresponding MICs of the reference peptide BP76. P9-4 (KWRRWIRWL), our newly designed nine-amino acid analogue, also has particularly low MICs of 3.1-6.2 μg ml⁻¹ against four of the tested pathogens; these MICs are two to eight times lower than those reported for Pac-525 (6.2-50 μg ml⁻¹).These new peptides (P11-5, P11-6 and P9-4) also exhibit improved stability in the presence of salts, and have low cytotoxicity as shown by the hemolysis and MTT assays. From the results of field-emission scanning electron microscopy, membrane depolarization and dye-leakage assays, we propose that these peptides exert their action by disrupting membrane lipids. Molecular dynamics simulation studies confirm that P11-6 peptide maintains relatively stable helical structure and exerts more perturbation action on the order of acyl tail of lipid bilayer.     

Antimicrobial peptides: key components of the innate immune system

Life-threatening infectious diseases are on their way to cause a worldwide crisis, as treating them effectively is becoming increasingly difficult due to the emergence of antibiotic resistant strains. Antimicrobial peptides (AMPs) form an ancient type of innate immunity found universally in all living organisms, providing a principal first-line of defense against the invading pathogens. The unique diverse function and architecture of AMPs has attracted considerable attention by scientists, both in terms of understanding the basic biology of the innate immune system, and as a tool in the design of molecular templates for new anti-infective drugs. AMPs are gene-encoded short (<100 amino acids), amphipathic molecules with hydrophobic and cationic amino acids arranged spatially, which exhibit broad spectrum antimicrobial activity. AMPs have been the subject of natural evolution, as have the microbes, for hundreds of millions of years. Despite this long history of co-evolution, AMPs have not lost their ability to kill or inhibit the microbes totally, nor have the microbes learnt to avoid the lethal punch of AMPs. AMPs therefore have potential to provide an important breakthrough and form the basis for a new class of antibiotics. In this review, we would like to give an overview of cationic antimicrobial peptides, origin, structure, functions, and mode of action of AMPs, which are highly expressed and found in humans, as well as a brief discussion about widely abundant, well characterized AMPs in mammals, in addition to pharmaceutical aspects and the additional functions of AMPs.     

Discovery and characterization of novel cyclotides originated from chimeric precursors consisting of albumin-1 chain a and cyclotide domains in the Fabaceae family

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1-T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants.     

Parallel evaluation of antimicrobial peptides derived from the synthetic PAF26 and the human LL37

The antimicrobial hexapeptide PAF26 was de novo designed towards phytopathogenic fungi of agricultural importance. To analyze its clinical potential, the activity of PAF26 has been determined against several microorganisms of clinical relevance including Staphylococcus, Candida, and several dermatophytes. For comparison purposes, the peptides KR20 and KI26 derived from the human cathelicidin LL37 were selected and fungal pathogens of agronomic relevance were included. PAF26 has similar antimicrobial activity in vitro compared to KR20 despite their different lengths and amino acid compositions. Moreover, neither peptide is lytic to human erythrocytes or keratinocytes. The hybrid peptide PAF26:KR20 showed better antimicrobial properties than the original peptides against most of the pathogens tested. The structural properties of PAF26:KR20 compared to related 26-amino acid peptides support the idea that the increment in toxicity correlates with positive charge and hydrophobicity. However, the degree of peptide helicity was not a predictor of antimicrobial activity.     

Fungal (-like) biocontrol organisms in tomato disease control

The worldwide important crop tomato is attacked by various pathogens, for which management is still primarily reliant on fungicides despite increasing concerns and constraints on their use. Other approaches are investigated, including the use of biocontrol organisms to manage tomato diseases. In this review we discuss and compare the interaction of major biocontrol fungi (BCF) with tomato, including the endophytic arbuscular mycorrhizal fungi and Piriformospora indica, the free-living opportunistic symbionts Trichoderma spp. and non-pathogenic Fusarium oxysporum, as well as the oomycete Pythium oligandrum. We cover recent advances that have been made in unraveling biocontrol modes of action against the most important tomato pathogens, encompassing direct effects of the BCF on pathogens and their indirect effects through the plant, with a main focus on induced systemic resistance. It is an exciting era for the study of biocontrol tripartite interactions, with the emergence of next-generation sequencing tools and the higher pace at which new genomes are being sequenced nowadays, as was recently also achieved for tomato. In addition, plant pathology and biocontrol research domains are increasingly reaching out to each other, because of the parallels that we are only beginning to discover between the interactions of beneficial and detrimental micro-organisms with a plant. Considering the enormous technological possibilities at hand today, this seems a timely opportunity to review the most recent advances in this field and to anticipate to what is ahead of us, discussing breakthroughs expected in our understanding of biocontrol interactions and remaining hurdles on the way to reach them.     

Antimicrobial defence and persistent infection in insects revisited

Insects show long-lasting antimicrobial immune responses that follow the initial fast-acting cellular processes. These immune responses are discussed to provide a form of phrophylaxis and/or to serve as a safety measure against persisting infections. The duration and components of such long-lasting responses have rarely been studied in detail, a necessary prerequisite to understand their adaptive value. Here, we present a 21 day proteomic time course of the mealworm beetle Tenebrio molitor immune-challenged with heat-killed Staphylococcus aureus. The most upregulated peptides are antimicrobial peptides (AMPs), many of which are still highly abundant 21 days after infection. The identified AMPs included toll and imd-mediated AMPs, a significant number of which have no known function against S. aureus or other Gram-positive bacteria. The proteome reflects the selective arena for bacterial infections. The results also corroborate the notion of synergistic interactions in vivo that are difficult to model in vitro.This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.     

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 μM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 μM. In combination, as little as 1.25 μM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics.     

Membrane-active antimicrobial peptide identified in Rana arvalis by targeted DNA sequencing

Antimicrobial peptides (AMPs) are naturally produced, gene encoded molecules with a direct antimicrobial activity against pathogens, often also showing other immune-related properties. Anuran skin secretions are rich in bioactive peptides, including AMPs, and we have reported a novel targeted sequencing approach to identify novel AMPs simultaneously in different frog species, from small quantities of skin tissue. Over a hundred full-length peptides were identified from specimens belonging to five different Ranidae frog species, out of which 29 were novel sequences. Six of these were selected for synthesis and testing against a panel of Gram-negative and Gram-positive bacteria. One peptide, identified in Rana arvalis, proved to be a potent and broad-spectrum antimicrobial, active against ATCC bacterial strains and a multi-drug resistant clinical isolate. CD spectroscopy suggests it has a helical conformation, while surface plasmon resonance (SPR) that it may self-aggregate/oligomerize at the membrane surface. It was found to disrupt the bacterial membrane at sub-MIC, MIC and above-MIC concentrations, as observed by flow cytometry and/or visualized by atomic force microscopy (AFM). Only a limited toxicity was observed towards peripheral blood mononuclear cells (PBMC) with a more pronounced effect observed against the MEC-1 cell line.