Search Results (10)

Background/Objectives: Currently, infections caused by fungi of the Candida genus remain a significant global health concern. The rising incidence of mycoses, coupled with the rapid emergence of fungal resistance, highlights the urgent need to search for new antifungal agents. Here, we obtained the recombinant hevein-like peptide from Amaranthus caudatus with two amino acid substitutions (F18W in the chitin-binding motif and M13A preventing the peptide from cleavage with cyanogen bromide during its biotechnological production). Methods: Antifungal potential of the modified hevein-like peptide, designated as mAc-AMP2, against susceptible and resistant strains of Candida albicans and non-albicans Candida species was studied. Results: We showed that mAc-AMP2 possessed anticandidal activities against all strains tested at nanomolar peptide concentrations. The presence of salts or serum affected the action of the peptide but its antifungal activity remained quite high. mAc-AMP2 exhibited anti-adherent properties and inhibited the formation of fungal biofilms. Using RP-HPLC, we demonstrated that degradation of the peptide in the presence of serum occurred rather slowly. mAc-AMP2 did not exhibit hemolytic and cytotoxic activities against the Caco-2 cell monolayer and peripheral blood mononuclear cells. Using flow cytometry, we demonstrated that the peptide at its high concentrations increased fungal membrane permeability. In resistance induction experiments, sensitivity of C. albicans toward mAc-AMP2 decreased over time, but restored after the peptide elimination. Conclusions: Taking into account all the data obtained, we suggest that the modified hevein-like peptide is a promising candidate for development of novel therapeutic agents to combat fungal infections caused by C. albicans and other Candida species. Full article

Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a–c and cL1–6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg²⁺ and Fe³⁺ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins. Full article

Antimicrobial peptides (AMPs) are short polypeptide molecules produced by multicellular organisms that are involved in host defense and microbiome preservation. In recent years, AMPs have attracted attention as novel drug candidates. However, their successful use requires detailed knowledge of the mode of action and identification of the determinants of biological activity. In this review, we focused on structure-function relationships in the thionins, α-hairpinins, hevein-like peptides, and the unique Ib-AMP peptides isolated from Impatiens balsamina. We summarized the available data on the amino acid sequences and 3D structure of peptides, their biosynthesis, and their biological activity. Special attention was paid to the determination of residues that play a key role in the activity and the identification of the minimal active cores. We have shown that even subtle changes in amino acid sequences can affect the biological activity of AMPs, which opens up the possibility of creating molecules with improved properties, better therapeutic efficacy, and cheaper large-scale production. Full article

Chitin-binding hevein-like peptides (CB-HLPs) belong to a family of cysteine-rich peptides that play important roles in plant stress and defense mechanisms. CB-HLPs are ribosomally synthesized peptides that are known to be bioprocessed from the following two types of three-domain CB-HLP precursor architectures: cargo-carrying and non-cargo-carrying. Here, we report the identification and characterization of chenotides biosynthesized from the third type of precursors, which are cleavable hololectins of the quinoa (Chenopodium quinoa) family. Chenotides are 6-Cys-CB-HLPs of 29–31 amino acids, which have a third type of precursor architecture that encompasses a canonical chitin-binding domain that is involved in chitin binding and anti-fungal activities. Microbroth dilution assays and microscopic analyses showed that chenotides are effective against phyto-pathogenic fungi in the micromolar range. Structure determination revealed that chenotides are cystine knotted and highly compact, which could confer resistance against heat and proteolytic degradation. Importantly, chenotides are connected by a novel 18-residue Gly/Ala-rich linker that is a target for bioprocessing by cathepsin-like endopeptidases. Taken together, our findings reveal that chenotides are a new family of CB-HLPs from quinoa that are synthesized as a single multi-modular unit and bioprocessed to yield individual mature CB-HLPs. Importantly, such precursors constitute a new family of cleavable hololectins. This unusual feature could increase the biosynthetic efficiency of anti-fungal CB-HLPs, to provide an evolutionary advantage for plant survival and reproduction. Full article

In the common chickweed Stellaria media, two antimicrobial peptides (AMPs), SmAMP1.1a and SmAMP1.2a, have been shown to be proteolytically released as products of the expression of a single gene, proSmAMP1. In this study, the gene proSmAMP1 was introduced into two potato varieties, Zhukovsky ranny and Udacha. These early-maturing varieties were shown to be susceptible to early blight caused by Alternaria spp. Most transgenic lines of either variety having strong expression of the target gene demonstrated high levels of resistance to Alternaria spp. during three years of cultivation, but did not otherwise differ from the initial varieties. Disease severity index (DSI) was introduced as a complex measure of plant susceptibility to early blight, taking into account the diameter of lesions caused by the Alternaria spp., the fungus sporulation intensity and its incubation period duration. Across all transgenic lines, the DSI inversely correlated both with the target gene expression and the copy number in the plant genome. Our results are promising for improving the resistance of potato and other crops to early blight by expression of AMPs from wild plants. Full article

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato. Full article

There are increasing environmental risks associated with extensive use of fungicides for crop protection. Hence, the use of new approaches using natural plant defense mechanisms, including application of plant antimicrobial peptides (AMPs), is of great interest. Recently, we studied the structural–function relationships between antifungal activity and five hevein-like AMPs from the WAMP (wheat AMP) family of Triticum kiharae Dorof. et Migush. We first discovered that short peptides derived from the central, N-, and C-terminal regions of one of the WAMPs (WAMP-2) were able to augment the inhibitory effect of Folicur^® EC 250, a triazole fungicide, on spore germination of the wheat pathogenic fungi, including Fusarium spp. and Alternaria alternata. In this research, we explored the ability of chemically synthesized WAMP-2-derived peptides for enhancing the sensitivity of two other Fusarium and Alternaria species, F. oxysporum and A. solani, causing wilt and early blight of tomato, respectively, to Folicur^®. The synthesized WAMP-2-derived peptides synergistically interacted with the fungicide and significantly increased its efficacy, inhibiting conidial germination at much lower Folicur^® concentrations than required for the same efficiency using the fungicide alone. The experiments on co-applications of some of WAMP-2-fragments and the fungicide on tomato leaves and seedlings, which confirmed the results obtained in vitro, are described. Full article

Hevein-like antimicrobial peptides (AMPs) comprise a family of plant AMPs with antifungal activity, which harbor a chitin-binding site involved in interactions with chitin of fungal cell walls. However, the mode of action of hevein-like AMPs remains poorly understood. This work reports the structure–function relationship in WAMPs—hevein-like AMPs found in wheat (Triticum kiharae Dorof. et Migush.) and later in other Poaceae species. The effect of WAMP homologues differing at position 34 and the antifungal activity of peptide fragments derived from the central, N- and C-terminal regions of one of the WAMPs, namely WAMP-2, on spore germination of different plant pathogenic fungi were studied. Additionally, the ability of WAMP-2-derived peptides to potentiate the fungicidal effect of tebuconazole, one of the triazole fungicides, towards five cereal-damaging fungi was explored in vitro by co-application of WAMP-2 fragments with Folicur^® EC 250 (25% tebuconazole). The antifungal activity of WAMP homologues and WAMP-2-derived peptides varied depending on the fungus, suggesting multiple modes of action for WAMPs against diverse pathogens. Folicur^® combined with the WAMP-2 fragments inhibited the spore germination at a much greater level than the fungicide alone, and the type of interactions was either synergistic or additive, depending on the target fungus and concentration combinations of the compounds. The combinations, which resulted in synergism and drastically enhanced the sensitivity to tebuconazole, were revealed for all five fungi by a checkerboard assay. The ability to synergistically interact with a fungicide and exacerbate the sensitivity of plant pathogenic fungi to a commercial antifungal agent is a novel and previously uninvestigated property of hevein-like AMPs. Full article

Cereal-derived bioactive peptides with antimicrobial activity have been poorly explored compared to those from dicotyledonous plants. Furthermore, there are a few reports addressing the structural differences between antimicrobial peptides (AMPs) from cultivated and wild cereals, which may shed light on significant varieties in the range and level of their antimicrobial activity. We performed a primary structure analysis of some antimicrobial peptides from wild and cultivated cereals to find out the features that are associated with the much higher antimicrobial resistance characteristic of wild plants. In this review, we identified and analyzed the main parameters determining significant antifungal activity. They relate to a high variability level in the sequences of C-terminal fragments and a high content of hydrophobic amino acid residues in the biologically active defensins in wild cereals, in contrast to AMPs from cultivated forms that usually exhibit weak, if any, activity. We analyzed the similarity of various physicochemical parameters between thionins and defensins. The presence of a high divergence on a fixed part of any polypeptide that is close to defensins could be a determining factor. For all of the currently known hevein-like peptides of cereals, we can say that the determining factor in this regard is the structure of the chitin-binding domain, and in particular, amino acid residues that are not directly involved in intermolecular interaction with chitin. The analysis of amino acid sequences of alpha-hairpinins (hairpin-like peptides) demonstrated much higher antifungal activity and more specificity of the peptides from wild cereals compared with those from wheat and corn, which may be associated with the presence of a mini cluster of positively charged amino acid residues. In addition, at least one hydrophobic residue may be responsible for binding to the components of fungal cell membranes. Full article

Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. Full article