Subinhibitory effects of 2,4-diacetylphloroglucinol on filamentous fungus Aspergillus fumigatus.

AIMS: Aspergillus fungi are common members of the soil microbiota. Some physiological and structural characteristics of Aspergillus species make them important participants in soil ecological processes. In this study, we aimed to evaluate the impact of 2,4-diacetylphloroglucinol (2,4-DAPG), a common metabolite of soil and rhizosphere bacteria, on the physiology of Aspergillus fumigatus. METHODS AND RESULTS: Integrated analysis using microscopy, spectrophotometry, and liquid chromatography showed the following effects of 2,4-DAPG on Aspergillus physiology. It was found that A. fumigatus in the biofilm state is resistant to high concentrations of 2,4-DAPG. However, experimental exposure led to a depletion of the extracellular polymeric substance, changes in the structure of the cell wall of the mycelium (increase in the content of α- and ß-glucans, chitin, and ergosterol), and conidia (decrease in the content of DHN-melanin). 2,4-DAPG significantly reduced the production of mycotoxins (gliotoxin and fumagillin) but increased the secretion of proteases and galactosaminogalactan. CONCLUSIONS: Overall, the data obtained suggest that 2,4-DAPG-producing Pseudomonas bacteria are unlikely to directly eliminate A. fumigatus fungi, as they exhibit a high level of resistance when in the biofilm state. However, at low concentrations, 2,4-DAPG significantly alters the physiology of aspergilli, potentially reducing the adaptive and competitive capabilities of these fungi.

Gausemycin A-Resistant Staphylococcus aureus Demonstrates Affected Cell Membrane and Cell Wall Homeostasis.

Antibiotic resistance is a significant and pressing issue in the medical field, as numerous strains of infectious bacteria have become resistant to commonly prescribed antibiotics. Staphylococcus aureus is a bacterium that poses a grave threat, as it is responsible for a large number of nosocomial infections and has high mortality rates worldwide. Gausemycin A is a new lipoglycopeptide antibiotic that has considerable efficacy against multidrug-resistant S. aureus strains. Although the cellular targets of gausemycin A have been previously identified, detailing the molecular processes of action is still needed. We performed gene expression analysis to identify molecular mechanisms that may be involved in bacterial resistance to gausemycin A. In the present study, we observed that gausemycin A-resistant S. aureus in the late-exponential phase showed an increased expression of genes involved in cell wall turnover (sceD), membrane charge (dltA), phospholipid metabolism (pgsA), the two-component stress-response system (vraS), and the Clp proteolytic system (clpX). The increased expression of these genes implies that changes in the cell wall and cell membrane are essential for the bacterial resistance to gausemycin A. In the stationary phase, we observed a decrease in the expression of genes involved in the phospholipid metabolism (mprF) and Clp proteolytic system (clpX).

Exploring the antibacterial action of gliotoxin: Does it induce oxidative stress or protein damage?

The study aimed to investigate the effects of gliotoxin (GTX), a secondary fungal metabolite belonging to the epipolythiodioxopiperazines class, on Gram-positive and Gram-negative bacteria. While the cytotoxic mechanism of GTX on eukaryotes is well understood, its interaction with bacteria is not yet fully comprehended. The study discovered that S. epidermidis displayed a higher uptake rate of GTX than E.coli. However, Gram-negative bacteria required higher doses of GTX than Gram-positive bacteria to experience the bactericidal effect, which occurred within 4 h for both types of bacteria. The treatment of bioluminescent sensor E.coli MG1655 pKatG-lux with GTX resulted in oxidative stress. Pre-incubation with the antioxidant Trolox did not increase the GTX inhibitory dose, however, slightly increased the bacterial growth rate comparing to GTX alone. At the same time, we found that GTX inhibitory dose was significantly increased by the pretreatment of bacteria with 2-mercaptoethanol and reduced glutathione. Using another biosensor, E. coli MG1655 pIpbA-lux, we showed that bacteria treated with GTX exhibited heat shock stress. SDS-page electrophoresis demonstrated protein aggregation under the GTX treatment. In addition, we have found that gliotoxin's action on bacteria was significantly inhibited when zinc salt was added to the growth medium.

Triazoles and Strobilurin Mixture Affects Soil Microbial Community and Incidences of Wheat Diseases.

Pesticides are widely used in agriculture as a pest control strategy. Despite the benefits of pesticides on crop yields, the persistence of chemical residues in soil has an unintended impact on non-targeted microorganisms. In the present study, we evaluated the potential adverse effects of a mixture of fungicides (difenoconazole, epoxiconazole, and kresoxim-methyl) on soil fungal and bacterial communities, as well as the manifestation of wheat diseases. In the fungicide-treated soil, the Shannon indices of both fungal and bacterial communities decreased, whereas the Chao1 indices did not differ compared to the control soil. Among bacterial taxa, the relative abundances of Arthrobacter and Sphingomonas increased in fungicide-treated soil due to their ability to utilize fungicides and other toxic compounds. Rhizopus and plant-beneficial Chaetomium were the dominant fungal genera, with their prevalence increasing by 2-4 times in the fungicide-treated soil. The genus Fusarium, which includes phytopathogenic species, which are notably responsible for root rot, was the most abundant taxon in each of the two conditions but its relative abundance was two times lower in fungicide-treated soils, consistent with a lower level of disease incidence in plants. The prediction of metabolic pathways revealed that the soil bacterial community had a high potential for degrading various pollutants, and the soil fungal community was in a state of recovery after the application of quinone outside inhibitor (QoI) fungicides. Fungicide-treated soil was characterized by an increase in soil microbial carbon, compared with the control soil. Collectively, the obtained results suggest that the application of difenoconazole, epoxiconazole, and kresoxim-methyl is an effective approach for pest control that does not pose a hazard for the soil ecosystem in the short term. However, it is necessary to carry out additional sampling to take into account the spatio-temporal impact of this fungicide mixture on the functional properties of the soil.

Staphylococcus aureus is able to generate resistance to novel lipoglycopeptide antibiotic gausemycin A.

Gausemycin A is the first member of the novel lipoglycopeptides family produced by Streptomyces roseoflavus INA-Ac-5812. Gausemycin A has a pronounced bactericidal activity against methicillin-resistant Staphylococcus aureus. However, the ability of S. aureus to be resistant to gausemycin A has not been investigated yet. Using serial passaging, we have obtained the resistant variant S. aureus 5812R, which is 80 times more resistant compared to the parent strain. Susceptibility testing of S. aureus 5812R revealed the acquisition of cross-resistance to daptomycin, cefazolin, tetracycline, and gentamicin, while the resistance to vancomycin, nisin, and ramoplanin was absent. Whole genome sequencing revealed single nucleotide polymorphism (SNP) and deletions in S. aureus 5812R, among which are genes encoding efflux pump (sepA), the two-component Kdp system (kdpE), and the component of isoprenoid biosynthesis pathway (hepT). Phenotypically, S. aureus 5812R resembles a small-colony variant, as it is slow-growing, forms small colonies, and is deficient in pigments. Profiling of fatty acids (FA) composition constituting the cytoplasmic membrane of S. aureus 5812R revealed the prevalence of anteiso-branched FA, while straight FA was slightly less present. The evidence also showed that the gausemycin A-resistant strain has increased expression of the cls2 gene of the cardiolipin synthase. The performed checkerboard assay pointed out that the combination of gausemycin A and ciprofloxacin showed a synergistic effect against S. aureus 5812R.

2,4-Diacetylphloroglucinol Modulates Candida albicans Virulence.

The dimorphic fungus Candida albicans is one of the most important opportunistic pathogens for humankind. The use of fungicides against Candida could be associated with sub-inhibitory effects, which are referred to as fungal stress responses and are undesirable for the host. In this work, we investigated the antifungal action of 2,4-diacetylphloroglucinol (2,4-DAPG) against Candida albicans ATCC 10231 with a focus on their biofilm-forming ability. We found that 2,4-DAPG was able to reduce the ability of Candida cells to form biofilms, but complete inhibition and eradication effects were not achieved. Furthermore, C. albicans cells in the adherent state were characterized by reduced susceptibility to 2,4-DAPG compared to planktonic cells. The investigation of the mechanisms that could explain the antibiofilm action of 2,4-DAPG revealed a reduction in the cell`s surface hydrophobicity and the inhibition of the yeast-to-hyphae transition. The inhibition of the Candida cells filamentation was accompanied by an increase in the expression of the NRG1 gene, which is a negative regulator of hyphal development. In addition, we microscopically visualized the treated biofilms and revealed numerous channels that were decorated with particles and localized on the hyphae. We assumed that these hyphal structures could be associated with the secretion of aspartyl proteases (Sap). The performed assessments revealed an increase in the activity of Sap, which was accompanied by an increase in the expression of the sap2 and sap4 genes. The antifungal action of 2,4-DAPG is known to be associated with affecting the permeability of cellular structures, which leads to H+ATPase malfunction and the disruption of mitochondrial respiration. The subsequent cytosol acidification and generation of ROS trigger the inhibition of Candida filamentation and activation of Sap production. The introduction of antioxidant Trolox simultaneously with 2,4-DAPG leads to a reduction in Sap production. Collectively, the obtained data indicate new aspects of the interaction of fungal cells with 2,4-DAPG, an antimicrobial metabolite of Pseudomonas spp.

Oak bark (Quercus sp. cortex) protects plants through the inhibition of quorum sensing mediated virulence of Pectobacterium carotovorum.

Bacterial intercellular communication mediated by small diffusible molecules, known as quorum sensing (QS), is a common mechanism for regulating bacterial colonisation strategies and survival. Influence on QS by plant-derived molecules is proposed as a strategy for combating phytopathogens by modulating their virulence. This work builds upon other studies that have revealed plant-derived QS inhibitors extracted from oak bark (Quercus sp.). It was found that co-incubation of Pectobacterium carotovorum VKM-B-1247 with oak bark extract (OBE) reduced the production of acyl-HSL. This was accompanied by a dose-dependent decrease in the bacterial cellulolytic and protease activity. At the transcriptomic level, the OBE treatment suppressed the main QS-related genes expR/expI. Potato tubers pre-treated with OBE showed resistance to a manifestation of soft-rot symptoms. Analysis of the component composition of the OBE identified several biologically active molecules, such as n-hexadecanoic acid, 2,6-di-tert-butyl-4-methylphenol, butylated hydroxytoluene (BHT), gamma-sitosterol, lupeol, and others. Molecular docking of the binding energy between identified molecules and homology models of LuxR-LuxI type proteins allow to identify potential inhibitors. Collectively, obtained results figure out great potential of widely distributed oak-derived plant material for bacterial control during storage of potato.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

Smart and Sustainable Nanotechnological Solutions in a Battle against COVID-19 and Beyond: A Critical Review.

The variety of available biocidal features make nanomaterials promising for fighting infections. To effectively battle COVID-19, categorized as a pandemic by the World Health Organization (WHO), materials scientists and biotechnologists need to combine their knowledge to develop efficient antiviral nanomaterials. By design, nanostructured materials (spherical, two-dimensional, hybrid) can express a diverse bioactivity and unique combination of specific, nonspecific, and mixed mechanisms of antiviral action. It can be related to the material's specific features and their multiple functionalization strategies. This is a complex guiding approach in which an interaction target is constantly moving and quickly changing. On the other hand, in such a rush, sustainability may be put aside. Therefore, to elucidate the most promising nanotechnological solutions, we critically review available data within the frame of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other types of viruses. We highlight solutions that are, or could be, more sustainable and less toxic. In this regard, reduction of the number of synthetic routes, organic solvents, byproducts, and residues is highly recommended. Such efficient, green solutions may be further used for the prevention of virion-host interactions, treatment of the already developed infection, reducing inflammation, and finally, protecting healthcare professionals with masks, fabrics, equipment, and in other associated areas. Further translation into the market needs putting on the fast track with respect to principles of green chemistry, feasibility, safety, and the environment.

Bacterial-Derived Plant Protection Metabolite 2,4-Diacetylphloroglucinol: Effects on Bacterial Cells at Inhibitory and Subinhibitory Concentrations.

2,4-Diacetylphloroglucinol (2,4-DAPG) is a well-known bacterial secondary metabolite, however, its mechanism of inhibitory and subinhibitory action on bacterial cells is still poorly understood. The mechanism of 2,4-DAPG action on model bacterial strains was investigated using fluorescent spectroscopy and the action of the antibiotic was found to involve a rapid increase in membrane permeability that was accompanied by a reduction in its viability in nutrient-poor medium. At the same time, antibacterial action in nutrient-rich medium developed for several hours. Atomic force microscopy demonstrated time-dependent disturbances in the outer membrane of Escherichia coli when exposed to 2,4-DAPG, while Staphylococcusaureus cells have been visualized with signs of intracellular leakage. In addition, 2,4-DAPG inhibited the metabolic activity of S. aureus and E. coli bacterial cells in mature biofilms. Observed differences in the antibiofilm activity were dependent upon antibiotic concentration. The intracellular targets of the action of 2,4-DAPG were assessed using bacterial biosensors with inducible bioluminescence corresponding to DNA and protein damage. It was unable to register any positive response from either sensor. As a result, the bactericidal action of 2,4-DAPG is believed to be associated with the destruction of the bacterial barrier structures. The subinhibitory effect of 2,4-diacetylphloroglucinol was tested on quorum-sensing mediated processes in Pectobacterium carotovorum. Subinhibitory concentrations of 2,4-DAPG were found to lower the biosynthesis of acyl-homoserine lactones in P. carotovorum in a dose-dependent manner. Further investigation elucidated that 2,4-DAPG inhibits the metabolic activity of bacteria without affecting their viability.

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.

A Novel Peptide Antibiotic Produced by Streptomyces roseoflavus Strain INA-Ac-5812 With Directed Activity Against Gram-Positive Bacteria.

In this work, we report the isolation and detailed functional characterization for the new non-ribosomally synthesized antibiotic 5812-A/C, which was derived from metabolites of Streptomyces roseoflavus INA-Ac-5812. According to its chemical structure, the studied 5812-A/C preliminary is composed of a cyclic peptide part covalently bounded with an arabinose residue. N-terminal amino acid sequencing of the native peptide has identified its partial structure of Leu-Asp-Gly-Ser-Gly and consisting of a Tyr residue that is supposed to have a two-component peptide nature for the molecule studied. However, the structural analysis of the antibiotic complex derived from S. roseoflavus INA-Ac-5812 is still ongoing. The mechanism of action of 5812-A/C was assessed in comparison with its most related analog, the lipopeptide antibiotic daptomycin, given the presence in both antimicrobials of an L-kynurenine amino acid residue. The inhibitory activity of 5812-A/C against Gram-positive bacteria including methicillin-resistant strain of Staphylococcus aureus was similar to daptomycin. The mechanism of action of 5812-A/C was associated with the disruption of membrane integrity, which differs in comparison with daptomycin and is most similar to the antimicrobial membrane-disturbing peptides. However, 5812-A/C demonstrated a calcium-dependent mode of action. In addition, unlike daptomycin, 5812-A/C was able to penetrate mature biofilms and inhibit the metabolic activity of embedded S. aureus cells. At the same time, 5812-A/C has no hemolytic activity toward erythrocyte, but possessed weak cytotoxic activity represented by heterochromatin condensation in human buccal epithelium cells. The biological properties of the peptide 5812-A/C suggest its classification as a calcium-dependent antibiotic effective against a wide spectrum of Gram-positive pathogenic bacteria.

Engineering of 2D Ti3C2 MXene Surface Charge and Its Influence on Biological Properties.

Current trends in the field of MXenes emphasize the importance of controlling their surface features for successful application in biotechnological areas. The ability to stabilize the surface properties of MXenes has been demonstrated here through surface charge engineering. It was thus determined how changing the surface charges of two-dimensional (2D) Ti3C2 MXene phase flakes using cationic polymeric poly-L-lysine (PLL) molecules affects the colloidal and biological properties of the resulting hybrid 2D nanomaterial. Electrostatic adsorption of PLL on the surface of delaminated 2D Ti3C2 flakes occurs efficiently, leads to changing an MXene's negative surface charge toward a positive value, which can also be effectively managed through pH changes. Analysis of bioactive properties revealed additional antibacterial functionality of the developed 2D Ti3C2/PLL MXene flakes concerning Escherichia. coli Gram-negative bacteria cells. A reduction of two orders of magnitude of viable cells was achieved at a concentration of 200 mg L-1. The in vitro analysis also showed lowered toxicity in the concentration range up to 375 mg L-1. The presented study demonstrates a feasible approach to control surface properties of 2D Ti3C2 MXene flakes through surface charge engineering which was also verified in vitro for usage in biotechnology or nanomedicine applications.

Sub-inhibitory Effects of Antimicrobial Peptides.

Antimicrobials, and particularly antimicrobial peptides (AMPs), have been thoroughly studied due to their therapeutic potential. The research on their exact mode of action on bacterial cells, especially at under sublethal concentrations, has resulted in a better understanding of the unpredictable nature of bacterial behavior under stress conditions. In this review, we were aiming to gather the wide yet still under-investigated knowledge about various AMPs and their subinhibition effects on cellular and molecular levels. We describe how AMP action is non-linear and unpredictable, also showing that exposure to AMP can lead to antimicrobial resistance via triggering various regulatory systems. Being one of the most known types of antimicrobials, bacteriocins have dual action and can also be utilized by microorganisms as signaling molecules at naturally achievable sub-inhibitory concentrations. The unpredictable nature of AMP action and the pathogenic response triggered by them remains an area of knowledge that requires further investigation.

Pore-forming bacteriocins: structural-functional relationships.

Peptides and proteins are important bioorganic compounds in nature, among which a special place is occupied by antimicrobial substances. There are more than 2000 different antimicrobial peptides (AMPs) produced by a variety of living organisms. Bacteriocins produced by bacteria are the minor group, whose chemical structures are most complicated among all AMPs. The review summarized the main points related to antimicrobial action of the bacteriocins including steps of peptide's interaction with bacterial membranes and details of membrane damaging. The membrane-disordered bacteriocins were described in accordance with structural-functional relationships.

A Novel Lipopeptaibol Emericellipsin A with Antimicrobial and Antitumor Activity Produced by the Extremophilic Fungus Emericellopsis alkalina.

Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. Additionally, emericellipsin A showed a significant cytotoxic effect and was highly active against Hep G2 and HeLa tumor cell lines. We used NMR spectroscopy to reveal that this peptaibol is nine amino acid residues long and contains non-standard amino acids. The mode of molecular action of emericellipsin A is most likely associated with its effects on the membranes of cells. Emericellipsin A is rather short peptaibol and could be useful for the development of antifungal, antibacterial, or anti-tumor remedies.

Genome Sequence of Enterococcus faecium Strain ICIS 96 Demonstrating Intermicrobial Antagonism Associated with Bacteriocin Production.

We report here the complete genome sequence of Enterococcus faecium strain ICIS 96, which was isolated from the feces of a horse. Bacteriological characterization of strain ICIS 96 revealed the absence of pathogenicity factors, while its spectrum of antagonistic activity was found to be broad, having activities associated with both Gram-positive and Gram-negative bacteria. Analysis of the E. faecium ICIS 96 genome revealed five genes associated with antimicrobial activity (enterocin [ent] A, ent B, lactobin A/cerein 7b, and ent L50 A/B). No genes that correlate with human pathogenicity were identified.

Purification of a Novel Bacteriocin-Like Inhibitory Substance Produced by Enterococcus faecium ICIS 8 and Characterization of Its Mode of Action.

OBJECTIVE: The aim of this work was to purify and characterize a bacteriocin-like antimicrobial substance produced by an antagonistic active strain of Enterococcus faecium. METHODS AND RESULTS: A novel bacteriocin-like inhibitory substance (BLIS) produced by the E. faecium ICIS 8 strain was purified and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and N-terminal amino acid sequencing revealed the following partial sequence: NH2-APKEKCFPKYCV. The proteinaceous nature of purified BLIS was assessed by treatment with proteolytic enzyme. Studies of the action of BLIS using bacteriological and bioluminescence assays revealed a dose-dependent inhibition of Listeria monocytogenes 88BK and Escherichia coli K12 TG1 lac::lux viability. The interaction of the BLIS with the bacterial surface led to the compensation of a negative charge value, as shown by zeta-potential measurements. Assessments of membrane integrity using fluorescent probes and atomic force microscopy revealed the permeabilization of the cellular barrier structures in both L. monocytogenes and E. coli. CONCLUSION: The novel BLIS from E. faecium ICIS 8 was characterized by a unique primary peptide sequence and exerted bactericidal activity against L. monocytogenes and E. coli by disrupting membrane integrity.

Studying of cellular interaction of hairpin-like peptide EcAMP1 from barnyard grass (Echinochloa crusgalli L.) seeds with plant pathogenic fungus Fusarium solani using microscopy techniques.

An interaction of recombinant hairpin-like cationic peptide EcAMP1 with conidia of plant pathogenic fungus Fusarium solani at the cellular level was studied by a combination of microscopic methods. EcAMP1 is from barnyard grass (Echinochloa crusgalli L.), and obtained by heterologous expression in Escherichia coli system. As a result, a direct relationship between hyphal growth inhibition and increasing active peptide concentration, time of incubation and fungal physiological condition has been determined. Dynamics of accumulation and redistribution of the peptide studied on fungal cellular cover and inside the conidia cells has been shown. The dynamics are dependent on time of coupling, as well as, a dissimilarity of EcAMP1 binding with cover of fungal conidia and its stepwise accumulation and diffuse localization in the cytoplasm. Correlation between structural disruption of fungal conidia and the presence of morphological changes has also been found. The correlation was found under the influence of peptide high concentrations at concentrations above 32 µM. The results indicate the presence of a binding of EcAMP1 with the surface of fungal conidia, thus, demonstrating a main specificity for its antifungal action at the cellular level. These results, however, cannot exclude the existence of attendant EcAMP1 action based on its intracellular localization on some specific targets. SCANNING 38:591-598, 2016. © 2016 Wiley Periodicals, Inc.