Svetlana Kryštofová

The kingdom of Fungi is rich in species that live in various environments and exhibit different lifestyles. Many are beneficial and indispensable for the environment and industries, but some can threaten plants, animals, and humans as pathogens. Various strategies have been applied to eliminate fungal pathogens by relying on chemical and nonchemical antifungal agents and tools. Nonthermal plasma (NTP) is a potential tool to inactivate pathogenic and food-contaminating fungi and genetically improve fungal strains used in industry as enzyme and metabolite producers. The NTP mode of action is due to many highly reactive species and their interactions with biological molecules. The interaction of the NTP with living cells is believed to be synergistic yet not well understood. This review aims to summarize the current NTP designs, applications, and challenges that involve fungi, as well as provide brief descriptions of underlying mechanisms employed by fungi in interactions with the NTP components

Aspergillus ochraceus is a soil fungus known to produce ochratoxin A, a harmful secondary metabolite. Prevention and control of fungal pathogens mostly rely on chemical fungicides, which is one of the contributing factors in the emergence of the fungal resistance, hence novel methods for fungal eradication have been extensively researched. The cold atmospheric pressure (CAP) plasma generated in ambient air has been recently applied in microbial decontamination. Here we used the diffuse coplanar surface barrier discharge in inactivation of a toxigenic strain A. ochraceus. The plasma-treated conidia and mycelium exhibited morphological changes such as ruptures and desiccation. Mycelium dehydration and changes in the chemical composition of hyphal surface accompanied plasma treatment. The growth of 26 h old mycelia were significantly restricted after 30 s of plasma treatment. The conidial vitality declined 4 logs after 180 s of plasma exposure leading to almost complete decontamination. After shorter plasma treatment of conidia, the ochratoxin A (OTA) production increased at the early stage of cultivation, but the overall level was significantly reduced compared to untreated samples after longer cultivation. Our results indicated that the fungal growth and the OTA production were significantly changed by plasma treatment and underscored CAP plasma as a promising method in the decontamination of A. ochraceus without a risk to generate strains with increased OTA production.

Functional dissection of γ-aminobutyric acid metabolism in Neurospora crassa

Rapidly evolving cold atmospheric pressure plasma (CAPP)–based technology has been actively used not only in bioresearch but also in biotechnology, food safety and processing, agriculture, and medicine. High variability in plasma device configurations and electrode layouts has accelerated non-thermal plasma applications in treatment of various biomaterials and surfaces of all sizes. Mode of cold plasma action is likely associated with synergistic effect of biologically active plasma components, such as UV radiation or reactive species. CAPP has been employed in inactivation of viruses, to combat resistant microorganisms (antibiotic resistant bacteria, spores, biofilms, fungi) and tumors, to degrade toxins, to modify surfaces and their properties, to increase microbial production of compounds, and to facilitate wound healing, blood coagulation, and teeth whitening. The mini-review provides a brief overview of non-thermal plasma sources and recent achievements in biological sciences. We have also included pros and cons of CAPP technologies as well as future directions in biosciences and their respective industrial fields.

The effect of light on the binding of Ca²⁺ to mycelia and to cell walls isolated from aerial mycelia of three strains of Trichoderma spp. was studied. Two independent methods were used to measure the total Ca²⁺ content in mycelia and the Ca²⁺ bound to cell walls isolated from aerial mycelia. The results of these methods showed that the light-induced formation and maturation of conidia in Trichoderma spp. is accompanied by increased Ca²⁺ deposition in mycelia and cell walls. Moreover, the cultivation of Trichoderma atroviride F-534 in the presence of ⁴⁵Ca²⁺ under circadian illumination showed that radioactivity was exclusively localized in the light-induced conidial rings of aerial mycelia. The fluorescence microscopy of chlortetracycline-stained mycelia showed that the major fraction of Ca²⁺ was accumulated in conidia and fructification structures, or some intracellular compartments in T. atroviride F-534 grown under circadian illumination, while only a limited amount of Ca²⁺ was associated with hyphal surfaces. In addition, the study of ⁴⁵Ca²⁺ binding to cell walls revealed that T. atroviride F-534 displays both increased ⁴⁵Ca²⁺ binding capacity and elevated affinity to ⁴⁵Ca²⁺ binding upon illumination. The results indicate that conidia formation and (or) maturation is associated with changes in Ca²⁺ homeostasis.

The cold atmospheric-pressure plasma (CAPP) has become one of the recent effective decontamination technologies, but CAPP interactions with biological material remain the subject of many studies. The CAPP generates numerous types of particles and radiations that synergistically affect cells and tissues differently depending on their structure. In this study, we investigated the effect of CAPP generated by diffuse coplanar surface barrier discharge on hyphae of Aspergillus flavus. Hyphae underwent massive structural changes after plasma treatment. Scanning electron microscopy showed drying hyphae that were forming creases on the hyphal surface. ATR-FTIR analysis demonstrated an increase of signal intensity for C=O and C-O stretching vibrations indicating chemical changes in molecular structures located on hyphal surface. The increase in membrane permeability was detected by the fluorescent dye, propidium iodide. Biomass dry weight determination and increase in permeability indicated leakage of cell content and subsequent death. Disintegration of nuclei and DNA degradation confirmed cell death after plasma treatment. Damage of plasma membrane was related to lipoperoxidation that was determined by higher levels of thiobarbituric acid reactive species after plasma treatment. The CAPP treatment led to rise of intracellular ROS levels detected by fluorescent microscopy using 2′,7′-dichlorodihydrofluorescein diacetate. At the same time, antioxidant enzyme activities increased, and level of reduced glutathione decreased. The results in this study indicated that the CAPP treatment in A. flavus targeted both cell surface structures, cell wall, and plasma membrane, inflicting injury on hyphal cells which led to subsequent oxidative stress and finally cell death at higher CAPP doses.

Pancreatic lipase inhibitors, such as tetrahydrolipstatin (orlistat), are used in anti-obesity treatments. Orlistat is the only anti-obesity drug approved by the European Medicines Agency (EMA). The drug is synthesized by saturation of lipstatin, a β-lactone compound, isolated from Streptomyces toxytricini and S. virginiae. To identify producers of novel pancreatic lipase inhibitors or microbial strains with improved lipstatin production and higher chemical purity remains still a priority. In this study, a high-throughput screening method to identify Streptomyces strains producing potent pancreatic lipase inhibitors was established. The assay was optimized and validated using S. toxytricini NRRL 15443 and its mutants. Strains grew in 24-well titer plates. Lipstatin levels were assessed directly in culture medium at the end of cultivation by monitoring lipolytic activity in the presence of a chromogenic substrate, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 6-methylresorufin ester (DGGR). The lipase activity decreased in response to lipstatin production, and this was demonstrated by accumulation of red-purple methylresorufin, a product of DGGR digestion. The sensitivity of the assay was achieved by adding a lipase of high lipolytic activity and sensitivity to lipstatin to the reaction mixture. In the assay, the fungal lipase from Mucor javanicus was used as an alternative to the human pancreatic lipase. Many fungal lipases preserve high lipolytic activity in extreme conditions and are not colipase dependent. The assay proved to be reliable in differentiation of strains with high and low lipstatin productivity.

The fungus Trichoderma atroviride is a member of the genus Trichoderma to which belong many species known for high cellulase production, formation of various antibiotics, plant biocontrol and antagonistic activities against other fungi. Deletion of T. atroviride glutamate decarboxylase gene gad caused minor defects in germination, hyphal branching, slower growth and disruption of conidiation pattern. GABA can be used by fungi as a secondary carbon source and as a primary nitrogen source. We analyzed the effect of different nutrient compositions and environmental conditions (light and temperature) on growth and development of T. atroviride in strains defective in the functional GAD. The gad mutants grown on NH₄NO₃ as a sole carbon source grew slower and formed conidiation bands closer to each other which was clearly demonstrated during their cultivation in race tubes. The gad mutants exhibited slightly lower apical extension growth rate at the room temperature but their apical extension rate dropped significantly at 30 °C. Higher temperature had also inhibitory effect on gad mutant conidiation, whereas 30 °C seems optimal temperature for the parental strain. The optimal temperature for gad mutant conidiation was lower than in F534, about 25 °C.

Targeted genome editing using engineered nucleases such as ZFNs and TALENs has been rapidly replaced by the CRISPR/Cas9 (clustered, regulatory interspaced, short palindromic/ CRISPR-associated nuclease) system. CRISPR/Cas9 technology represents a significant improvement enabling a new level of targeting, efficiency and simplicity. Gene editing mediated by CRISPR/Cas9 has been recently used not only in bacteria but in many eukaryotic cells and organisms, from yeasts to mammals. Other modifications of the CRISPR-Cas9 system have been used to introduce heterologous domains to regulate gene expressions or label specific loci in various cell types. The review focuses not only on native CRISPR/Cas systems which evolved in prokaryotes as an endogenous adaptive defense mechanism against foreign DNA attacks, but also on the CRISPR/Cas9 adoption as a powerful tool for site-specific gene modifications in fungi, plants and mammals.

G protein−coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization.