Γ-core Motif Research Articles

The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens.

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and β-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Synergistic action of synthetic peptides and amphotericin B causes disruption of the plasma membrane and cell wall in Candida albicans.

The objective of this work was to evaluate the combination of synthetic peptides based on the γ-core motif of defensin PvD1 with amphotericin B (AmB) at different concentrations against Candida albicans. We applied the checkerboard assay using different concentrations of the commercial drug AmB and the synthetic peptides γ31-45PvD1++ and γ33-41PvD1++ against C. albicans, aiming to find combinations with synergistic interactions. Between these two interactions involving γ31-45PvD1++ and AmB, an additive effect was observed. One such interaction occurred at concentrations of 0.009 µM of peptide γ31-45PvD1++ and 13.23 µM of AmB and another condition of 0.019 µM of peptide γ31-45PvD1++ and 6.61 µM of AmB. The other two concentrations of the interaction showed a synergistic effect in the combination of synthetic peptide γ31-45PvD1++ and AmB, where the concentrations were 1.40 µM peptide γ31-45PvD1++ and 0.004 µM AmB and 0.70 µM γ31-45PvD1++ peptide and 0.002 µM AmB. We proceeded with analysis of the mechanism of action involving synergistic effects. This examination unveiled a range of impactful outcomes, including the impairment of mitochondrial functionality, compromise of cell wall integrity, DNA degradation, and a consequential decline in cell viability. We also observed that both synergistic combinations were capable of causing damage to the plasma membrane and cell wall, causing leakage of intracellular components. This discovery demonstrates for the first time that the synergistic combinations found between the synthetic peptide γ31-45PvD1++ and AmB have an antifungal effect against C. albicans, acting on the integrity of the plasma membrane and cell wall.

Rational Design of Antifungal Peptides Based on the γ-Core Motif of a Neosartorya (Aspergillus) fischeri Antifungal Protein to Improve Structural Integrity, Efficacy, and Spectrum.

Antifungal peptides offer promising alternative compounds for the treatment of fungal infections, for which new antifungal compounds are urgently needed. Constant and broad antifungal spectra of these peptides play essential roles in their reliable therapeutic application. It has been observed that rationally designed peptides using the evolutionarily conserved γ-core region (GXC-X3-9-C) of an antifungal protein from Neosartorya (Aspergillus) fischeri highly inhibit the growth of fungi. The cysteines in these peptides have free sulfhydryl groups, which allow cyclization and dimerization under oxidative conditions, thereby impairing antifungal efficacy. To overcome this problem, one or two cysteine residues were substituted by serines or S-tert-butyl was applied as a cysteine-protecting group. Furthermore, structural integrity and antifungal efficacy investigations before and after oxidative exposure revealed that substituting both cysteines with serines and S-tert-butylation helped maintain the structural integrity. However, it slightly decreased the antifungal efficacy against a yeast, Candida albicans. Interestingly, S-tert-butylation maintained the efficacy and could extend the antifungal activity to a mold, Aspergillus fumigatus. Usually, cyclization and dimerization did not influence the antifungal efficacy of most peptides. Additionally, hemolysis tests and Galleria mellonella toxicity model experiments indicated that none of the applied modifications made the peptides harmful to animals.

Small Cationic Cysteine-Rich Defensin-Derived Antifungal Peptide Controls White Mold in Soybean.

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.

γ-Core Guided Antibiotic Design Based on Human Enteric Defensin 5.

An increase in the number of infections caused by resistant bacteria worldwide necessitates the development of alternatives to antibiotics. Human defensin (HD) 5 is an innate immune peptide with broad-spectrum antibacterial activity, but its complicated structure makes its preparation difficult. Herein, we truncated the HD5 structure by extracting the highly conserved γ-core motif. A structure-activity study showed that this motif was ineffective in killing bacteria in the absence of specific spatial conformation. Notably, after the introduction of two intramolecular disulfide bonds, its antibacterial activity was markedly improved. Glu and Ser residues were then replaced with Arg to create the derivative RC18, which exhibited stronger potency than HD5, particularly against methicillin-resistant S. aureus (MRSA). Mechanistically, RC18 bound to lipid A and lipoteichoic acid at higher affinities than HD5. Furthermore, RC18 was more efficient than HD5 in penetrating the bacterial membranes. Molecular dynamics simulation revealed that five Arg residues, Arg1, Arg7, Arg9, Arg15, and Arg18, mediated most of the polar interactions of RC18 with the phospholipid head groups during membrane penetration. In vivo experiments indicated that RC18 decreased MRSA colonization and dramatically improved the survival of infected mice, thus demonstrating that RC18 is a promising drug candidate to treat MRSA infections.

The γ-Core Motif Peptides of Plant AMPs as Novel Antimicrobials for Medicine and Agriculture.

The γ-core motif is a structural element shared by most host antimicrobial peptides (AMPs), which is supposed to contribute to their antimicrobial properties. In this review, we summarized the available data on the γ-core peptides of plant AMPs. We describe γ-core peptides that have been shown to exhibit inhibitory activity against plant and human bacterial and fungal pathogens that make them attractive scaffolds for the development of novel anti-infective agents. Their advantages include origin from natural AMP sequences, broad-spectrum and potent inhibitory activity, and cost-effective production. In addition, some γ-core peptides combine antimicrobial and immunomodulatory functions, thus broadening the spectrum of practical applications. Some act synergistically with antimycotics and fungicides, so combinations of peptides with conventionally used antifungal agents can be suggested as an effective strategy to reduce the doses of potentially harmful chemicals. The presented information will pave the way for the design of novel antimicrobials on the basis of γ-core motif peptides, which can find application in medicine and the protection of crops from diseases.

The γ-Core Motif Peptides of AMPs from Grasses Display Inhibitory Activity against Human and Plant Pathogens.

Antimicrobial peptides (AMPs) constitute an essential part of the plant immune system. They are regarded as alternatives to conventional antibiotics and pesticides. In this study, we have identified the γ-core motifs, which are associated with antimicrobial activity, in 18 AMPs from grasses and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. All the tested peptides displayed antimicrobial properties. We discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides designed from the 4-Cys-containing defensin-like peptides, whose role in plant immunity has remained unknown, as well as the knottin-like peptide and the C-terminal prodomain of the thionin, which points to the direct involvement of these peptides in defense mechanisms. Studies of the mode of action of the eight most active γ-core motif peptides on yeast cells using staining with propidium iodide showed that all of them induced membrane permeabilization leading to cell lysis. In addition to identification of the antimicrobial determinants in plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to control plant pathogens.

Structure-Activity Predictions From Computational Mining of Protein Databases to Assist Modular Design of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are naturally produced by pro- and eukaryotes and are promising alternatives to antibiotics to fight multidrug-resistant microorganisms. However, despite thousands of AMP entries in respective databases, predictions about their structure–activity relationships are still limited. Similarly, common or dissimilar properties of AMPs that have evolved in different taxonomic groups are nearly unknown. We leveraged data entries for 10,987 peptides currently listed in the three antimicrobial peptide databases APD, DRAMP and DBAASP to aid structure–activity predictions. However, this number reduced to 3,828 AMPs that we could use for computational analyses, due to our stringent quality control criteria. The analysis uncovered a strong bias towards AMPs isolated from amphibians (1,391), whereas only 35 AMPs originate from fungi (0.9%), hindering evolutionary analyses on the origin and phylogenetic relationship of AMPs. The majority (62%) of the 3,828 AMPs consists of less than 40 amino acids but with a molecular weight higher than 2.5 kDa, has a net positive charge and shares a hydrophobic character. They are enriched in glycine, lysine and cysteine but are depleted in glutamate, aspartate and methionine when compared with a peptide set of the same size randomly selected from the UniProt database. The AMPs that deviate from this pattern (38%) can be found in different taxonomic groups, in particular in Gram-negative bacteria. Remarkably, the γ-core motif claimed so far as a unifying structural signature in cysteine-stabilised AMPs is absent in nearly 90% of the peptides, questioning its relevance as a prerequisite for antimicrobial activity. The disclosure of AMPs pattern and their variation in producing organism groups extends our knowledge of the structural diversity of AMPs and will assist future peptide screens in unexplored microorganisms. Structural design of peptide antibiotic drugs will benefit using natural AMPs as lead compounds. However, a reliable and statistically balanced database is missing which leads to a large knowledge gap in the AMP field. Thus, thorough evaluation of the available data, mitigation of biases and standardised experimental setups need to be implemented to leverage the full potential of AMPs for drug development programmes in the clinics and agriculture.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical β-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

Antifungal Potency and Modes of Action of a Novel Olive Tree Defensin Against Closely Related Ascomycete Fungal Pathogens.

Antimicrobial peptides play a pivotal role in the innate immunity of plants. Defensins are cysteine-rich antifungal peptides with multiple modes of action. A novel Oleaceae-specific defensin gene family has been discovered in the genome sequences of wild and cultivated species of a perennial olive tree, Olea europaea. OefDef1.1, a member of this defensin family, potently inhibits the in-vitro growth of ascomycete fungal pathogens Botrytis cinerea and three Fusarium spp. OefDef1.1 rapidly permeabilizes the plasma membrane of the conidial and germling cells of B. cinerea. Interestingly, it induces reactive oxygen species and translocates to the cytoplasm only in the germlings but not in the conidia. In medium containing a high concentration of Na1+, antifungal activity of OefDef1.1 is significantly reduced. Surprisingly, a chimeric OefDef1.1 peptide containing the γ-core motif of a Medicago truncatula defensin, MtDef4, displays Na1+-tolerant antifungal activity. In a phospholipid-protein overlay assay, the chimeric peptide exhibits stronger binding to its phosphoinositide partners than OefDef1.1 and is also more potent in inhibiting gray mold disease on the surface of Nicotiana benthamiana and lettuce leaves than OefDef1.1. Significant differences are observed among the four ascomycete pathogens in their responses to OefDef1.1 in growth medium with or without the elevated concentration of Na1+. The varied responses of closely related ascomycete pathogens to this defensin have implications for engineering disease resistance in plants.

Worms' Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms. In metazoans, they act as host defense factors by eliminating microbial pathogens. But they also help to select the colonizing bacterial symbionts while coping with specific environmental challenges. Although many AMPs share common structural characteristics, for example having an overall size between 10–100 amino acids, a net positive charge, a γ-core motif, or a high content of cysteines, they greatly differ in coding sequences as a consequence of multiple parallel evolution in the face of pathogens. The majority of AMPs is specific of certain taxa or even typifying species. This is especially the case of annelids (ringed worms). Even in regions with extreme environmental conditions (polar, hydrothermal, abyssal, polluted, etc.), worms have colonized all habitats on Earth and dominated in biomass most of them while co-occurring with a large number and variety of bacteria. This review surveys the different structures and functions of AMPs that have been so far encountered in annelids and nematodes. It highlights the wide diversity of AMP primary structures and their originality that presumably mimics the highly diverse life styles and ecology of worms. From the unique system that represents marine annelids, we have studied the effect of abiotic pressures on the selection of AMPs and demonstrated the promising sources of antibiotics that they could constitute.

Gene isolation and structural characterization of a legume tree defensin with a broad spectrum of antimicrobial activity.

The recombinant EcgDf1 defensin has an antimicrobial effect against both plant and human pathogens. In silico analyses predict that EcgDf1 is prone to form dimers capable of interacting with the membranes of microorganisms. Plant defensins comprise a large family of antimicrobial peptides (AMP) with a wide range of biological functions. They are cysteine-rich molecules, highly sequence diverse but with a conserved and stable structure. In this work, a defensin gene (EcgDf1) was isolated from Erythrina crista-galli, a legume tree native from South America. The predicted peptide presents eight cysteines, with a γ-core motif GXCX3-9C and six cysteines distributed like the typical defensin αβ motif. The mature EcgDf1 coding sequence was heterologously expressed in Escherichia coli strains and purified by affinity chromatography. Possible dimer and oligomers of EcgDf1 were visible in SDS electrophoresis. Moreover, its 3D structure, determined by homology modeling, docking, and molecular dynamics simulations, was found to be compatible with the formation of homodimers between the β3 and β1-loop-α1, leaving the β2-loop-β3 free to interact with lipid membranes. The purified recombinant peptide inhibited the growth of several critical plant and human pathogens, like the opportunistic fungi Candida albicans and Aspergillus niger and the plant pathogens Clavibacter michiganensis ssp. michiganensis, Penicillium expansum, Botrytis cinerea, and Alternaria alternata. EcgDf1 is a promising candidate for the development of antimicrobial products for use in agriculture and medicine.

Antibacterial and antifungal activity of defensins from the Australian paralysis tick, Ixodes holocyclus

Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 – 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1–4 (HoloTickCores 1–4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 μM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.

Plant Defensin Peptides have Antifungal and Antibacterial Activity Against Human and Plant Pathogens.

Plant defensins are small, cysteine-rich antimicrobial peptides. These peptides have previously been shown to primarily inhibit the growth of fungal plant pathogens. Plant defensins have a γ-core motif, defined as GXCX3-9C, which is required for their antifungal activity. To evaluate plant defensins as a potential control for a problematic agricultural disease (alfalfa crown rot), short, chemically synthesized peptides containing γ-core motif sequences were screened for activity against numerous crown rot pathogens. These peptides showed both antifungal and, surprisingly, antibacterial activity. Core motif peptides from Medicago truncatula defensins (MtDef4 and MtDef5) displayed high activity against both plant and human bacterial pathogens in vitro. Full-length defensins had higher antimicrobial activity compared with the peptides containing their predictive γ-core motifs. These results show the future promise for controlling a wide array of economically important fungal and bacterial plant pathogens through the transgenic expression of a plant defensin. They also suggest that plant defensins may be an untapped reservoir for development of therapeutic compounds for combating human and animal pathogens.

Improved smallest peptides based on positive charge increase of the γ-core motif from PνD1 and their mechanism of action against Candida species.

BackgroundPlant defensins have a hallmark γ-core motif (GXCX3-9C) that is related to their antimicrobial properties. The aim of this work was to design synthetic peptides based on the region corresponding to the PvD1 defensin γ-core that are the smallest amino acid sequences that bear the strongest biological activity.MethodsWe made rational substitutions of negatively charged amino acid residues with positively charged ones, and the reduction in length in the selected PvD1 γ-core sequence to verify whether the increased net positive charges and shortened length are related to the increase in antifungal activity. Herein, we opted to evaluate the action mechanism of γ33-41PvD1++ peptide due to its significant inhibitory effect on tested yeasts. In addition, it is the smallest construct comprising only nine amino acid residues, giving it a better possibility to be a prototype for designing a new antifungal drug, with lower costs to the pharmaceutical industry while still maintaining the strongest antimicrobial properties.ResultsThe γ33-41PvD1++ peptide caused the most toxic effects in the yeast Candida buinensis, leading to membrane permeabilization, viability loss, endogenous reactive oxygen species increase, the activation of metacaspase, and the loss of mitochondrial functionality, suggesting that this peptide triggers cell death via apoptosis.ConclusionWe observed that the antifungal activity of PvD1 is not strictly localized in the structural domain, which comprises the γ-core region and that the increase in the net positive charge is directly related to the increase in antifungal activity.

A Computational Modeling Approach Predicts Interaction of the Antifungal Protein AFP from Aspergillus giganteus with Fungal Membranes via Its γ-Core Motif.

Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus, is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity.IMPORTANCE Fungal pathogens pose a serious danger to human welfare since they kill more people per year than malaria or tuberculosis and are responsible for crop losses worldwide. The treatment of fungal infections is becoming more complicated as fungi develop resistances against commonly used fungicides. Therefore, discovery and development of novel antifungal agents are of utmost importance.

Structural and biological features of a novel plant defensin from Brugmansia x candida.

Data from both the laboratory and clinic in the last decade indicate that antimicrobial peptides (AMPs) are widely regarded as potential sources of future antibiotics owing to their broad-spectrum activities, rapid killing, potentially low-resistance rate and multidirectional mechanisms of action compared to conventional antibiotics. Defensins, a prominent family of AMPs, have been found in a wide range of organisms including plants. Thailand is a rich source of plants including medicinal plants used therapeutically, however there is no report of defensin from among these plants. In this study, a novel plant defensin gene, BcDef, was successfully cloned from Brugmansia x candida (Bc). BcDef cDNA was 237 bp in length, encoding 78 amino acids with a putative 31-amino acid residue signal peptide at the N-terminal followed by the mature sequence. BcDef shared high sequence identity (78–85%) with Solanaceae defensins and belonged to the class I plant defensins. From homology modeling, BcDef shared a conserved triple stranded β-sheet (β1-β3) and one α-helix (α1) connected by a loop (L1-L3). BcDef1 peptide, designed from the γ-core motifs of BcDef located in loop 3, showed antibacterial activity against both Gram-positive and Gram-negative pathogens with the lowest MIC (15.70 μM) against Staphylococcus epidermidis. This peptide affected cell membrane potential and permeability, and caused cell membrane disruption. Moreover, BcDef1 also exhibited antioxidant activity and showed low cytotoxicity against mouse fibroblast L929 cells. These findings may provide an opportunity for developing a promising antibacterial agent for medical application in the future.

Rice Defensin OsAFP1 is a New Drug Candidate against Human Pathogenic Fungi

Fungal infections, such as candidiasis and aspergillosis, are some of the most frequent infections in humans. Although antifungal drugs are available for the treatment of these infections, antifungal agents with new mechanisms of action should be developed because of the increasing incidence of drug-resistant pathogens in recent years. In this study, a basic functional analysis of rice defensin OsAFP1, a novel antifungal drug candidate, was conducted. OsAFP1 exerted fungicidal activity against Candida albicans, the most common pathogenic fungus in humans, at 4 μM concentration, but it did not inhibit the growth of human pathogenic bacteria. In addition, OsAFP1 retained structural stability after heat treatment at 100 °C for 10 min and after serum treatment at 37 °C for 24 h. A propidium iodide (PI) uptake assay and mutational analysis revealed that amino acid residues within the C-terminal γ-core motif of OsAFP1, particularly Leu-39 and Lys-41, play an important role in its antifungal activity. Further, PI uptake and apoptosis assays suggested that OsAFP1 exerts its antifungal activity by inducing apoptosis of target cells. Immunohistochemistry showed that the OsAFP1 target molecule was located in the cell wall. These findings indicate that OsAFP1 may be developed into a potent antifungal drug.