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.

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.

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.

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.

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.