Editor’s Choice Articles

The mitochondrial electron transport chain consists of the classical protein complexes (I–IV) that facilitate the flow of electrons and coupled oxidative phosphorylation to produce metabolic energy. The canonical route of electron transport may diverge by the presence of alternative components to the electron transport chain. The following study comprises the bioinformatic identification and functional characterization of a putative alternative oxidase in the smut fungus Sporisorium reilianum f. sp. zeae. This alternative respiratory component has been previously identified in other eukaryotes and is essential for alternative respiration as a response to environmental and chemical stressors, as well as for developmental transitionaoxs during the life cycle of an organism. A growth inhibition assay, using specific mitochondrial inhibitors, functionally confirmed the presence of an antimycin-resistant/salicylhydroxamic acid (SHAM)-sensitive alternative oxidase in the respirasome of S. reilianum. Gene disruption experiments revealed that this enzyme is involved in the pathogenic stage of the fungus, with its absence effectively reducing overall disease incidence in infected maize plants. Furthermore, gene expression analysis revealed that alternative oxidase plays a prominent role in the teliospore developmental stage, in agreement with favoring alternative respiration during quiescent stages of an organism’s life cycle. Full article

The boletoid genera Butyriboletus and Exsudoporus have recently been suggested by some researchers to constitute a single genus, and Exsudoporus was merged into Butyriboletus as a later synonym. However, no convincing arguments have yet provided significant evidence for this congeneric placement. In this study, we analyze material from Exsudoporus species and closely related taxa to assess taxonomic and phylogenetic boundaries between these genera and to clarify species delimitation within Exsudoporus. Outcomes from a multilocus phylogenetic analysis (ITS, nrLSU, tef1-α and rpb2) clearly resolve Exsudoporus as a monophyletic, homogenous and independent genus that is sister to Butyriboletus. An accurate morphological description, comprehensive sampling, type studies, line drawings and a historical overview on the nomenclatural issues of the type species E. permagnificus are provided. Furthermore, this species is documented for the first time from Israel in association with Quercus calliprinos. The previously described North American species Exsudoporus frostii and E. floridanus are molecularly confirmed as representatives of Exsudoporus, and E. floridanus is epitypified. The eastern Asian species Leccinum rubrum is assigned here to Exsudoporus based on molecular evidence, and a new combination is proposed. Sequence data from the original material of the Japanese Boletus kermesinus were generated, and its conspecificity with L. rubrum is inferred as formerly presumed based on morphology. Four additional cryptic species from North and Central America previously misdetermined as either B. frostii or B. floridanus are phylogenetically placed but remain undescribed due to the paucity of available material. Boletus weberi (syn. B. pseudofrostii) and Xerocomus cf. mcrobbii cluster outside of Exsudoporus and are herein assigned to the recently described genus Amoenoboletus. Biogeographic distribution patterns are elucidated, and a dichotomous key to all known species of Exsudoporus worldwide is presented. Full article

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery. Full article

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of sixteen plasma membrane carboxylate transporters belonging to the AceTr, SHS, TDT, MCT, SSS, and DASS families was performed by heterologous expression in Saccharomyces cerevisiae. The newly identified C. jadinii transporters present specificity for mono-, di-, and tricarboxylates. The transporters CjAto5, CjJen6, CjSlc5, and CjSlc13-1 display the broadest substrate specificity; CjAto2 accepts mono- and dicarboxylates; and CjAto1,3,4, CjJen1-5, CjSlc16, and CjSlc13-2 are specific for monocarboxylic acids. A detailed characterization of these transporters, including phylogenetic reconstruction, 3D structure prediction, and molecular docking analysis is presented here. The properties presented by these transporters make them interesting targets to be explored as organic acid exporters in microbial cell factories. Full article

Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products. Full article

Candida albicans (CA), a commensal and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract and causes life-threatening infections. Antibiotic-induced gut dysbiosis is a major risk factor for increased CA colonization and dissemination from the GI tract. We identified a significant increase of taurocholic acid (TCA), a major bile acid in antibiotic-treated mice susceptible to CA infection. In vivo findings indicate that administration of TCA through drinking water is sufficient to induce colonization and dissemination of CA in wild-type and immunosuppressed mice. Treatment with TCA significantly reduced mRNA expression of immune genes ang4 and Cxcr3 in the colon. In addition, TCA significantly decreased the relative abundance of three culturable species of commensal bacteria, Turicibacter sanguinis, Lactobacillus johnsonii, and Clostridium celatum, in both cecal contents and mucosal scrapings from the colon. Taken together, our results indicate that TCA promotes fungal colonization and dissemination of CA from the GI tract by controlling the host defense system and intestinal microbiota that play a critical role in regulating CA in the intestine. Full article

Mucormycosis, a rare but highly fatal infection, is caused by fungi of the order Mucorales. Due to their ubiquitous nature, reduced susceptibility to antifungals, acid tolerance, and ability to infect immunocompromised patients through rapid dissemination, these fungi have been frequently reported to infect the COVID-19 patients. In order to develop strategies to overcome mucormycosis, it is essential to understand and identify novel Mucorales present in the environment. In this study, we report the identification of four novel pathogenic Mucorales using the silkworm (Bombyx mori) model. The strains’ phylogeny was analyzed using the genome sequence of the large subunit ribosomal ribonucleic acid (LSU rRNA) and the internal transcribed spacer (ITS) region, where strains 1-3, 5-3, and S286-1101 claded with Mucor orantomantidis, and strain 827-14 claded with Backusella lamprospora. All the strains had a cold-sensitive phenotype with their inability to grow prominently at 4 °C. Mucor sp. 1-3 and 5-3 were characterized by their filamentous and yeast-like growth under aerobic and anaerobic conditions, respectively. The yeast colonies of Mucor sp. 5-3 had multipolar budding cells often observed with cleaved cell surfaces under a scanning electron microscope. We further found that these strains were able to kill immunocompromised mice suggesting their pathogenicity to mammals. Our study established an invertebrate model-based screening system to identify novel pathogenic Mucorales from the natural environment and provided a clue towards the rapid increase in COVID-19 related mucormycosis. Full article

The contribution of fungi to the degradation of plant litter and transformation of dissolved organic matter (humic substances, in particular) in freshwater ecosystems has received increasing attention recently. However, the role of Saprolegniales as one of the most common eukaryotic organisms is rarely studied. In this study, we isolated and phylogenetically placed 51 fungal and 62 Saprolegniales strains from 12 German lakes. We studied the cellulo-, lignino-, and chitinolytic activity of the strains using plate assays. Furthermore, we determined the capacity of 10 selected strains to utilize 95 different labile compounds, using Biolog FF MicroPlates™. Finally, the ability of three selected strains to utilize maltose and degrade/produce humic substances was measured. Cladosporium and Penicillium were amongst the most prevalent fungal strains, while Saprolegnia, Achlya, and Leptolegnia were the most frequent Saprolegniales strains. Although the isolated strains assigned to genera were phylogenetically similar, their enzymatic activity and physiological profiling were quite diverse. Our results indicate that Saprolegniales, in contrast to fungi, lack ligninolytic activity and are not involved in the production/transformation of humic substances. We hypothesize that Saprolegniales and fungi might have complementary roles in interacting with dissolved organic matter, which has ecological implications for carbon cycling in freshwater ecosystems. Full article

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development. Full article

Zn₂Cys₆ transcription factors are unique to fungi and are involved in different regulatory functions. In this study, we have identified the Penicillium digitatumPdMut3 gene, which encodes a putative Zn (II) 2Cys6 DNA-binding protein. Elimination of PdMut3 in Pd1 strain caused increased virulence during citrus infection. The transcription of the PdMut3 gene showed a higher expression rate during fungal growth and less transcription during fruit infection. Furthermore, the deletion of the gene in the wild-type isolate of P. digitatum did not produce any modification of the sensitivity to different fungicides, indicating that the gene is not associated with resistance to fungicides. In contrast, PdMut3 null mutants showed a reduction in growth in minimal media, which was associated with severe alterations in conidiophore development and morphological alterations of the hyphae. Mutants showed greater sensitivity to compounds that interfere with the cell wall and an invasive growth block. Thus, PdMut3 might have an indirect role in fungi virulence through metabolism and peroxisomes development. Full article

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections. Full article

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s). Full article

Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is a transcriptional repressor for the expression of hyphal-specific genes in morphogenesis. Azoles or echinocandin drugs have been extensively prescribed for C. albicans infections, which has led to the development of drug-resistant strains. Therefore, it is necessary to develop new molecules to effectively treat fungal infections. Here, we showed that Molecule B and Molecule C, which contained a carbazole structure, attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that Molecule B and Molecule C inhibit morphogenesis through repressing protein and RNA levels of Ras/MAPK-related genes, including UME6 and NRG1. Furthermore, we determined the antifungal effects of Molecule B and Molecule C in vivo using a candidiasis murine model. We anticipate our findings are that Molecule B and Molecule C, which inhibits the Ras1/MAPK pathway, are promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases. Full article

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the belowground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root endosphere, the rhizosphere and bulk soil by internal transcribed spacer (ITS) high-throughput amplicon sequencing (HTAS). We imposed three irrigation regimes (100%, 50%, and 25% of the field capacity) to one-year old grapevine rootstock plants cv. SO₄ when plants had developed 2–3 roots. Root endosphere, rhizosphere, and bulk soil samples were collected 6- and 12-months post-plantation. Drought significantly modified the overall fungal composition of all three compartments, with the root endosphere compartment showing the greatest divergence from well-watered control (100%). The overall response of the fungal microbiota associated with black-foot disease (Dactylonectria and “Cylindrocarpon” genera) and the potential biocontrol agent Trichoderma to drought stress was consistent across compartments, namely that their relative abundances were significantly higher at 50–100% than at 25% irrigation regime. We identified a significant enrichment in several fungal genera such as the arbuscular mycorrhizal fungus Funneliformis during drought at 25% watering regime within the roots. Our results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in the restructuring of grapevine root microbial communities, and suggest the possibility that members of the altered grapevine microbiota might contribute to plant survival under extreme environmental conditions. Full article

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions. Full article

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus fumigatus antigens. Dual co-stimulation strongly enhanced A. fumigatus-induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an A. fumigatus lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with Aspergillus-associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens. Full article

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H₂O₂-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes. Full article

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent against fungal plant pathogens. The recognition and appropriate morphogenetic responses to prey-derived signals are essential for successful mycoparasitism. We established microcolony confrontation assays using T. atroviride strains expressing cell division cycle 42 (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1) interactive binding (CRIB) reporters to analyse morphogenetic changes and the dynamic displacement of localized GTPase activity during polarized tip growth. Microscopic analyses showed that Trichoderma experiences significant polarity stress when approaching its fungal preys. The perception of prey-derived signals is integrated via the guanosine triphosphatase (GTPase) and mitogen-activated protein kinase (MAPK) signalling network, and deletion of the MAP kinases Trichoderma MAPK 1 (Tmk1) and Tmk3 affected T. atroviride tip polarization, chemotropic growth, and contact-induced morphogenesis so severely that the establishment of mycoparasitism was highly inefficient to impossible. The responses varied depending on the prey species and the interaction stage, reflecting the high selectivity of the signalling process. Our data suggest that Tmk3 affects the polarity-stress adaptation process especially during the pre-contact phase, whereas Tmk1 regulates contact-induced morphogenesis at the early-contact phase. Neither Tmk1 nor Tmk3 loss-of-function could be fully compensated within the GTPase/MAPK signalling network underscoring the crucial importance of a sensitive polarized tip growth apparatus for successful mycoparasitism. Full article

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51–100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical–chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi. Full article

Early-diverging fungi harbour unprecedented diversity in terms of living forms, biological traits and genome architecture. Before the sequencing era, non-Dikarya fungi were considered unable to produce secondary metabolites (SM); however, this perspective is changing. The main classes of secondary metabolites in fungi include polyketides, nonribosomal peptides, terpenoids and siderophores that serve different biological roles, including iron chelation and plant growth promotion. The same classes of SM are reported for representatives of early-diverging fungal lineages. Encouraged by the advancement in the field, we carried out a systematic survey of SM in Mucoromycotina and corroborated the presence of various SM clusters (SMCs) within the phylum. Among the core findings, considerable representation of terpene and nonribosomal peptide synthetase (NRPS)-like candidate SMCs was found. Terpene clusters with diverse domain composition and potentially highly variable products dominated the landscape of candidate SMCs. A uniform low-copy distribution of siderophore clusters was observed among most assemblies. Mortierellomycotina are highlighted as the most potent SMC producers among the Mucoromycota and as a source of novel peptide products. SMC identification is dependent on gene model quality and can be successfully performed on a batch scale with genomes of different quality and completeness. Full article

Pregnant women have an increased risk of vulvovaginal candidosis. Recurrent candidosis is under debate as a contributor to preterm birth, and vertical transmission may cause diaper dermatitis and oral thrush in the newborn. Apart from cultural methods, the gold standard for diagnosing candidosis is Gram staining, which is time-consuming and requires laboratory facilities. The objective of this prospective study was to validate a point-of-care vaginal yeast detection assay (SavvyCheck™ Vaginal Yeast Test) and to evaluate it in asymptomatic pregnant women. We enrolled 200 participants, 100 of whom had vulvovaginal candidosis according to Gram stain (study group) and 100 were healthy pregnant controls (control group). Of these, 22 participants (11%) had invalid test results. The point-of-care test of the remaining 85 and 93 study participants in the study and control groups, respectively, showed a sensitivity of 94.1%, specificity of 98.9%, positive predictive value of 90.3%, and negative predictive value of 99.4% when compared with Gram stain. In conclusion, we found a high correlation between the SavvyCheck™ Vaginal Yeast Test and Gram-stained smears during pregnancy. This suggests a potential role of this point-of-care test as a screening tool for asymptomatic pregnant women in early gestation. Full article

An increase in fungal spores in ambient air is reported during a spike in particulate matter (PM_2.5 and PM₁₀) aerosols generated during dust or smog events. However, little is known about the impact of ambient bioaerosols on fungal infections in humans. To identify the correlation between the incidence of pulmonary aspergillosis and PM-associated bioaerosols (PM_2.5 and PM₁₀), we retrospectively analyzed data between 2015 and 2018 (first stage) and prospectively analyzed data in 2019 (second stage). Patient data were collected from patients in three medical institutions in Tainan, a city with a population of 1.88 million, located in southern Taiwan. PM data were obtained from the Taiwan Air Quality Monitoring Network. Overall, 544 non-repeated aspergillosis patients (first stage, n = 340; second stage, n = 204) were identified and enrolled for analysis. The trend of aspergillosis significantly increased from 2015 to 2019. Influenza A (H1N1) and ambient PMs (PM_2.5 and PM₁₀) levels had significant effects on aspergillosis from 2015 to 2018. However, ambient PMs and influenza A (H1N1) in Tainan were correlated with the occurrence of aspergillosis in 2018 and 2019, respectively. Overall (2015–2019), aspergillosis was significantly correlated with influenza (p = 0.002), influenza A (H1N1) (p < 0.001), and PM_2.5 (p = 0.040) in Tainan City. Using a stepwise regression model, influenza A (H1N1) (p < 0.0001) and Tainan PM₁₀ (p = 0.016) could significantly predict the occurrence of aspergillosis in Tainan. PM-related bioaerosols and influenza A (H1N1) contribute to the incidence of pulmonary aspergillosis. Full article

A new ectomycorrhizal species was discovered during the first survey of fungal diversity at Brijuni National Park (Croatia), which consists of 14 islands and islets. The National Park is located in the Mediterranean Biogeographical Region, a prominent climate change hot-spot. Inocybe brijunica sp. nov., from sect. Hysterices (Agaricales, Inocybaceae), is described based on morphology and multilocus phylogenetic data. The holotype collection was found at the edge between grassland and Quercus ilex forest with a few planted Pinus pinea trees, on Veli Brijun Island, the largest island of the archipelago. It is easily recognized by a conspicuous orange to orange–red–brown membranaceous surface layer located at or just above the basal part of the stipe. Other distinctive features of I. brijunica are the medium brown, radially fibrillose to rimose pileus; pale to medium brown stipe with fugacious cortina; relatively small, amygdaliform to phaseoliform, and smooth basidiospores, measuring ca. 6.5–9 × 4–5.5 µm; thick-walled, utriform, lageniform or fusiform pleurocystidia (lamprocystidia) with crystals and mostly not yellowing in alkaline solutions; cheilocystidia of two types (lamprocystidia and leptocystidia); and the presence of abundant caulocystidia only in the upper 2–3 mm of the stipe. Phylogenetic reconstruction of a concatenated dataset of the internal transcribed spacer region (ITS), the nuclear 28S rRNA gene (nrLSU), and the second largest subunit of RNA polymerase II (rpb2) resolved I. brijunica and I. glabripes as sister species. Full article

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems. Full article

This study was designed to analyze the interaction of 21 antifungal combinations consisting of seven major antifungal agents against 11 echinocandin- susceptible and six-resistant C. glabrata isolates. The combinations were divided into five major groups and were evaluated by checkerboard, disc diffusion, and time-killing assays. Synergy based on the fractional inhibitory concentration index of ≤0.50 was observed in 17.65–29.41% of the cases for caspofungin combinations with azoles or amphotericin B. Amphotericin B combination with azoles induced synergistic interaction in a range of 11.76–29.41%. Azole combinations and 5-flucytosine combinations with azoles or amphotericin B did not show synergistic interactions. None of the 21 combinations showed antagonistic interactions. Interestingly, 90% of the detected synergism was among the echinocandin-resistant isolates. Disk diffusion assays showed that the inhibition zones produced by antifungal combinations were equal to or greater than those produced by single drugs. The time-killing assay showed the synergistic action of caspofungin combination with fluconazole, voriconazole, and posaconazole, and the amphotericin B-5-flucytosine combination. Furthermore, for the first time, this assay confirmed the fungicidal activity of caspofungin-voriconazole and amphotericin B-5-flucytosine combinations. The combination interactions ranged from synergism to indifference and, most importantly, no antagonism was reported and most of the synergistic action was among echinocandin-resistant isolates. Full article

Methanol is a promising feedstock for metabolically competent yeast strains-based biorefineries. However, methanol toxicity can limit the productivity of these bioprocesses. Therefore, the identification of genes whose expression is required for maximum methanol tolerance is important for mechanistic insights and rational genomic manipulation to obtain more robust methylotrophic yeast strains. The present chemogenomic analysis was performed with this objective based on the screening of the Euroscarf Saccharomyces cerevisiae haploid deletion mutant collection to search for susceptibility phenotypes in YPD medium supplemented with 8% (v/v) methanol, at 35 °C, compared with an equivalent ethanol concentration (5.5% (v/v)). Around 400 methanol tolerance determinants were identified, 81 showing a marked phenotype. The clustering of the identified tolerance genes indicates an enrichment of functional categories in the methanol dataset not enriched in the ethanol dataset, such as chromatin remodeling, DNA repair and fatty acid biosynthesis. Several genes involved in DNA repair (eight RAD genes), identified as specific for methanol toxicity, were previously reported as tolerance determinants for formaldehyde, a methanol detoxification pathway intermediate. This study provides new valuable information on genes and potential regulatory networks involved in overcoming methanol toxicity. This knowledge is an important starting point for the improvement of methanol tolerance in yeasts capable of catabolizing and copying with methanol concentrations present in promising bioeconomy feedstocks, including industrial residues. Full article

Fungi and the mycobiome are a fundamental part of the human microbiome that contributes to human health and development. Despite this, relatively little is known about the mycobiome of the preterm infant gut. Here, we have characterised faecal fungal communities present in 11 premature infants born with differing degrees of prematurity and mapped how the mycobiome develops during early infancy. Using an ITS1 sequencing-based approach, the preterm infant gut mycobiome was found to be often dominated by a single species, typically a yeast. Candida was the most abundant genus, with the pathobionts C.albicans and C.parapsilosis highly prevalent and persistent in these infants. Gestational maturity at birth affected the distribution and abundance of these Candida, with hospital-associated C.parapsilosis more prevalent and abundant in infants born at less than 31 weeks. Fungal diversity was lowest at 6 months, but increased with age and change of diet, with food-associated Saccharomycescerevisiae most abundant in infants post weaning. This study provides a first insight into the fungal communities present within the preterm infant gut, identifying distinctive features including the prominence of pathobiont species, and the influence age and environmental factors play in shaping the development of the mycobiome. Full article