Recombinant Fusion Protein Containing Plant Nigellothionin Regulates the Growth of Food-Spoiling Fungus (Aspergillus niger).

This study aimed to obtain a recombinant chimeric protein named trx-NsW2 via theheterologous expression of the multifunctional antimicrobial peptide nigellothionin from black cumin (Nigella sativa L.) seeds in the Escherichia coli system. The protein was purified using a combination of Ni-NTA affinity chromatography and reversed-phase HPLC. Based on the HPLC calibration, the total yield of the protein was calculated to be 650 mg/L of bacterial culture. The fungistatic activity of trx-NsW2 against the food-spoiling fungus Aspergillus niger was demonstrated as itinhibited the maturation of conidiawithout affecting conidial germination or fungal growth. In contrast to mature nigellothionin NsW2, the fusion protein showeda low level of cytotoxicity towards both normal and tumor cell lines at concentrationsof up to 100-200 µM. Interestingly, at lower concentrations, it even stimulated cytokinesis. These findings are of critical importance for applying chimeric antimicrobial proteins obtained via microbiological synthesis in applied science.

Diversity of Cationic Antimicrobial Peptides in Black Cumin (Nigella sativa L.) Seeds.

Black cumin (Nigella sativa L.) is known to possess a wide variety of antimicrobial peptides belonging to different structural families. Three novel antimicrobial peptides have been isolated from black cumin seeds. Two of them were attributed as members of the non-specific lipid transfer proteins family, and one as a defensin. We have made an attempt of using the proteomic approach for novel antimicrobial peptides search in N. sativa seeds as well. The use of a well-established approach that includes extraction and fractionation stages remains relevant even in the case of novel peptides search because of the lacking N. sativa genome data. Novel peptides demonstrate a spectrum of antimicrobial activity against plant pathogenic organisms that may cause economically important crop diseases. These results obtained allow considering these molecules as candidates to be applied in "next-generation" biopesticides development for agricultural use.

C60 Fullerene Clusters Stabilize the Biologically Inactive Form of Topotecan.

There is a range of experimental proofs that biologically relevant compounds change their activity in the presence of C60 fullerene clusters in aqueous solution, which most frequently act as a nanoplatform for drug delivery. Inspired by this evidence, we made an effort to investigate the interaction of fullerene clusters with the antibiotic topotecan (TPT). This study proceeded in three steps, namely, UV/vis titration to confirm complexation and in vitro assays on proliferating and nonproliferating cells to elucidate the role of C60 fullerene in the putative change in TPT activity. Surprisingly, although the nonproliferating cell assay is consistent with the titration data and confirms complex formation, it contradicted the results of the proliferating cell assay. The latter showed that the mixture of TPT and fullerene affects the cells in the same way as pure TPT, as if there were no fullerenes in solution at all, whereas the action of TPT was expected to be enhanced. We explained this contradiction by the specific stabilization of the biologically inactive carboxylate form of the antibiotic adsorbed in the alkaline shell of large fullerene clusters, which leads to neutralization of the drug delivery function and almost zero net biological effect of the antibiotic in vitro. The practical outcome of the work is that fullerene clusters can be used for the selective delivery of pH-sensitive drug forms.

Rational Design of Plant Hairpin-like Peptide EcAMP1: Structural-Functional Correlations to Reveal Antibacterial and Antifungal Activity.

Plant antimicrobial peptides from the α-hairpinins family (hairpin-like peptides) are known to possess a wide range of biological activities. However, less is known about the structural determinants of their antimicrobial activity. Here, we suggest that spatial structure as well as surface charge and hydrophobicity level contribute to the antimicrobial properties of α-hairpinin EcAMP1 from barnyard grass (Echinochloa cruss-galli) seeds. To examine the role of the peptide spatial structure, two truncated forms of EcAMP1 restricted by inner and outer cysteine pairs were synthesized. It was shown that both truncated forms of EcAMP1 lost their antibacterial activity. In addition, their antifungal activity became weaker. To review the contribution of surface charge and hydrophobicity, another two peptides were designed. One of them carried single amino acid substitution from tryptophan to alanine residue at the 20th position. The second one represented a truncated form of the native EcAMP1 lacking six C-terminal residues. But the α-helix was kept intact. It was shown that the antifungal activity of both modified peptides weakened. Thereby we can conclude that the secondary structural integrity, hydrophobic properties, and surface charge all play roles in the antimicrobial properties of α-hairpinins. In addition, the antibacterial activity of cereal α-hairpinins against Gram-positive bacteria was described for the first time. This study expands on the knowledge of structure-function interactions in antimicrobial α-hairpinins.

Exploring Peptaibol's Profile, Antifungal, and Antitumor Activity of Emericellipsin A of Emericellopsis Species from Soda and Saline Soils.

Features of the biochemical adaptations of alkaliphilic fungi to exist in extreme environments could promote the production of active antibiotic compounds with the potential to control microorganisms, causing infections associated with health care. Thirty-eight alkaliphilic and alkalitolerant Emericellopsis strains (E. alkalina, E. cf. maritima, E. cf. terricola, Emericellopsis sp.) isolated from different saline soda soils and belonging to marine, terrestrial, and soda soil ecological clades were investigated for emericellipsin A (EmiA) biosynthesis, an antifungal peptaibol previously described for Emericellopsis alkalina. The analysis of the Emericellopsis sp. strains belonging to marine and terrestrial clades from chloride soils revealed another novel form with a mass of 1032.7 Da, defined by MALDI-TOF Ms/Ms spectrometers, as the EmiA lacked a hydroxyl (dEmiA). EmiA displayed strong inhibitory effects on cell proliferation and viability of HCT 116 cells in a dose- and time-dependent manners and induced apoptosis.

Nigellothionins from Black Cumin (Nigella sativa L.) Seeds Demonstrate Strong Antifungal and Cytotoxic Activity.

High-cationic biologically active peptides of the thionins family were isolated from black cumin (Nigella sativa L.) seeds. According to their physicochemical characteristics, they were classified as representatives of the class I thionin subfamily. Novel peptides were called "Nigellothionins", so-called because of their source plant. Thionins are described as components of plant innate immunity to environmental stress factors. Nine nigellothionins were identified in the plant in different amounts. Complete amino acid sequences were determined for three of them, and a high degree of similarity was detected. Three nigellothionins were examined for antifungal properties against collection strains. The dominant peptide, NsW2, was also examined for activity against clinical isolates of fungi. Cytotoxic activity was determined for NsW2. Nigellothionins activity against all collection strains and clinical isolates varied from absence to a value comparable to amphotericin B, which can be explained by the presence of amino acid substitutions in their sequences. Cytotoxic activity in vitro for NsW2 was detected at sub-micromolar concentrations. This has allowed us to propose an alteration of the molecular mechanism of action at different concentrations. The results obtained suggest that nigellothionins are natural compounds that can be used as antimycotic and anti-proliferative agents.

Peptide Extracts from Seven Medicinal Plants Discovered to Inhibit Oomycete Phytophthora infestans, a Causative Agent of Potato Late Blight Disease.

We report the inhibitory effect of peptide extracts obtained from seven medicinal plants against a causative agent of late blight disease Phytophthora infestans. We find that all the extracts possess inhibitory activity toward the zoospores output, zoosporangium germination, and the development of P. infestans on potato disc tubers at different quantitative levels. Based on the biological effects detected, an extract of common horsetail (Equisetum arvense) biomass is recognized as the most effective and is selected for further structural analysis. We perform a combination of amino acid analysis and MALDI-TOF mass spectrometry, which reveal the presence of Asn/Asp- and Gln/Glu-rich short peptides with molecular masses in the range of 500-900 Da and not exceeding 1500 Da as the maximum. Analytical anion-exchange HPLC is successfully applied for separation of the peptide extract from common horsetail (E. arvense). We collect nine dominant components that are combined in two groups with differences in retention times. The N-terminal amino acid sequence of the prevalent compounds after analytical ion-exchange HPLC allows us to identify them as peptide fragments of functionally active proteins associated with photosynthesis, aquatic transport, and chitin binding. The anti-oomycete effects may be associated with the conversion of ribulose-1,5-bisphosphate carboxylase/oxygenase to produce a number of biologically active anionic peptides with possible regulatory functions. These data inform our knowledge regarding biologically active peptide fragments; they are the components of programmed or induced proteolysis of plant proteins and can realize secondary antimicrobial functions.

Isolation of antimicrobial peptides from different plant sources: Does a general extraction method exist?

Plants are good sources of biologically active compounds with antimicrobial activity, including polypeptides. Antimicrobial peptides (AMPs) represent one of the main barriers of plant innate immunity to environmental stress factors and are attracting much research interest. There are some extraction methods for isolation of AMPs from plant organs based on the type of extractant and initial fractionation stages. But most methods are directed to obtain some specific structural types of AMPs and do not allow to understand the molecular diversity of AMP inside a whole plant. In this mini-review, we suggest an optimized scheme of AMP isolation from plants followed by obtaining a set of peptides belonging to various structural families. This approach can be performed for large-scale screening of plants to identify some novel or homologous AMPs for fundamental and applied studies.