Search Results (6)

Fungal mycelial pellets (MPs) exhibit high biomass-loading capacity; however, their application in wastewater treatment is constrained by structural fragility and the risk of environmental dispersion. To overcome these limitations, a dual-crosslinked polyvinyl alcohol–alginate gel (10% PVA, 2% sodium alginate) embedding strategy was developed and stabilized using 2% CaCl₂ and saturated boric acid. This encapsulation enhanced the tensile strength of MPs by 499% (310.4 vs. 62.1 kPa) and improved their settling velocity by 2.3-fold (1.12 vs. 0.49 cm/s), which was critical for stability under turbulent bioreactor conditions. Following encapsulation, the specific oxygen uptake rates (SOURs) of three fungal strains (F557, Y3, and F507) decreased by 30.3%, 54.8%, and 48.3%, respectively, while maintaining metabolic functionality. SEM revealed tight adhesion between the gel layer and both surface and internal hyphae, with the preservation of porous channels conducive to microbial colonization. In sequential-batch reactors treating sulfamethoxazole (SMX)-contaminated wastewater, gel-encapsulated MPs combined with acclimated sludge consistently achieved 72–75% SMX removal efficiency over six cycles, outperforming uncoated MPs (efficiency decreased from 81.2% to 58.7%) and pure gel–sludge composites (34–39%). The gel coating inhibited hyphal dispersion by over 90% and resisted mechanical disintegration under 24 h agitation. This approach offers a scalable and environmentally sustainable means of enhancing MPs’ operational stability in continuous-flow systems while mitigating fungal dissemination risks. Full article

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. Full article

Mitochondrial porin, the voltage-dependent anion-selective channel (VDAC), is the most abundant protein in the outer membrane, and is critical for the exchange of metabolites and phospholipids in yeast and mammals. However, the functions of porin in phytopathogenic fungi are not known. In this study, we characterized a yeast porin orthologue, Fgporin, in Fusarium graminearum. The deletion of Fgporin resulted in defects in hyphal growth, conidiation, and perithecia development. The Fgporin deletion mutant showed reduced virulence, deoxynivalenol production, and lipid droplet accumulation. In addition, the Fgporin deletion mutant exhibited morphological changes and the dysfunction of mitochondria, and also displayed impaired autophagy in the non-nitrogen medium compared to the wild type. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that Fgporin interacted with FgUps1/2, but not with FgMdm35. Taken together, these results suggest that Fgporin is involved in hyphal growth, asexual and sexual reproduction, virulence, and autophagy in F. graminearum. Full article

Voltage-dependent anion-selective channels (VDAC) maintain the bidirectional flow of small metabolites across the mitochondrial outer membrane and participate in the regulation of multiple cellular processes. To understand the roles of VDAC in cellular homeostasis, preliminary proteomic analyses of S100 cytosolic and mitochondria-enriched fractions from a VDAC-less Neurospora crassa strain (ΔPor-1) were performed. In the variant cells, less abundant proteins include subunits of translation initiation factor eIF-2, enzymes in the shikimate pathway leading to precursors of aromatic amino acids, and enzymes involved in sulfate assimilation and in the synthesis of methionine, cysteine, alanine, serine, and threonine. In contrast, some of the more abundant proteins are involved in electron flow, such as the α subunit of the electron transfer flavoprotein and lactate dehydrogenase, which is involved in one pathway leading to pyruvate synthesis. Increased levels of catalase and catalase activity support predicted increased levels of oxidative stress in ΔPor-1 cells, and higher levels of protein disulfide isomerase suggest activation of the unfolded protein response in the endoplasmic reticulum. ΔPor-1 cells are cold-sensitive, which led us to investigate the impact of the absence of VDAC on several mitochondrial membrane characteristics. Mitochondrial membranes in ΔPor-1 are more fluid than those of wild-type cells, the ratio of C18:1 to C18:3n3 acyl chains is reduced, and ergosterol levels are lower. In summary, these initial results indicate that VDAC-less N. crassa cells are characterized by a lower abundance of proteins involved in amino acid and protein synthesis and by increases in some associated with pyruvate metabolism and stress responses. Membrane lipids and hyphal morphology are also impacted by the absence of VDAC. Full article

In Candida albicans, calcium ions (Ca²⁺) regulate the activity of several signaling pathways, especially the calcineurin signaling pathway. Ca²⁺ homeostasis is also important for cell polarization, hyphal extension, and plays a role in contact sensing. It is therefore important to obtain accurate tools with which Ca²⁺ homeostasis can be addressed in this fungal pathogen. Aequorin from Aequorea victoria has been used in eukaryotic cells for detecting intracellular Ca²⁺. A codon-adapted aequorin Ca²⁺-sensing expression system was therefore designed for probing cytosolic Ca²⁺ flux in C. albicans. The availability of a novel water-soluble formulation of coelenterazine, which is required as a co-factor, made it possible to measure bioluminescence as a readout of intracellular Ca²⁺ levels in C. albicans. Alkaline stress resulted in an immediate influx of Ca²⁺ from the extracellular medium. This increase was exacerbated in a mutant lacking the vacuolar Ca²⁺ transporter VCX1, thus confirming its role in Ca²⁺ homeostasis. Using mutants in components of a principal Ca²⁺ channel (MID1, CCH1), the alkaline-dependent Ca²⁺ spike was greatly reduced, thus highlighting the crucial role of this channel complex in Ca²⁺ uptake and homeostasis. Exposure to the antiarrhythmic drug amiodarone, known to perturb Ca²⁺ trafficking, resulted in increased cytoplasmic Ca²⁺ within seconds that was abrogated by the chelation of Ca²⁺ in the external medium. Ca²⁺ import was also dependent on the Cch1/Mid1 Ca²⁺ channel in amiodarone-exposed cells. In conclusion, the aequorin Ca²⁺ sensing reporter developed here is an adequate tool with which Ca²⁺ homeostasis can be investigated in C. albicans. Full article

The paucity of effective antifungals against Aspergillus and increasing resistance, the recognition of the importance of Aspergillus biofilm in several clinical settings, and reports of verapamil—a calcium channel blocker—efficacy against Candida biofilm and hyphal growth, and synergy with an azole antifungal in vitro, led to a study of verapamil ± voriconazole against Aspergillus. Broth macrodilution methodology was utilized for MIC (minimum inhibitory concentration) and MFC (minimum fungicidal concentration) determination. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to verapamil, verapamil plus voriconazole, or voriconazole alone. For biofilm formation, we found less inhibition from the combinations than with either drug alone, or less inhibition from the combination than that of the more potent drug alone. For preformed biofilm, we found no significant change in activity comparing voriconazole alone compared to added verapamil, and no significant alteration of activity of the more potent voriconazole, at any concentration in the range tested, by addition of a concentration of verapamil that is inhibitory alone. In full checkerboard assays with planktonic fungus, there was no indication of any effect of one drug on the other (indifference). Although verapamil was similarly inactive against planktonic Aspergillus, as with Candida, verapamil was indeed active against Aspergillus biofilm. However, indifference and antagonism was found with voriconazole. Full article