Journal of Fungi

Aspergillus fumigatus is a world-wide saprophyte filamentous fungus which released conidia, its infectious morphotype, in the atmosphere. These conidia are inhaled daily by humans and can colonize the respiratory tract, where they may develop into hyphae, the invasive morphotype. We previously showed that bronchial epithelial cells (BECs) restrict A. fumigatus virulence by inhibiting conidial germination and filament formation through a process requiring PI3K signaling and the conidial fucose-specific lectin FleA. In the present study, we are looking to identify host factors and cellular partners involved in the BEC antifungal response and to define the molecular interactions underpinning FleA recognition. For this, we analyzed transcriptome of BECs infected with A. fumigatus in the presence or absence of the PI3K inhibitor LY294002. Functional involvement of candidate genes was assessed by siRNA knockdown and readouts of fungal filamentation (microscopic scoring and galactomannan release). FleA-interacting host proteins were identified by biotin-FleA affinity co-precipitation coupled to Tandem mass spectrometry, and validated by surface plasmon resonance and biolayer interferometry. The spatiotemporal dynamics of FleA and candidate partners were analyzed by confocal microscopy and proximity ligation assay We demonstrated that BEC antifungal activity involves at least two complementary pathways: a PI3K/laminin-332 axis promoting conidial adhesion, and a FleA-dependent pathway engaging ITGB1 and MRC2 consistent with lectin uptake and trafficking toward LAMP1-positive compartments. These findings nominate FleA-host receptor interactions as attractive targets for anti-adhesive strategies against A. fumigatus. Author summaryFungal pathogens are an increasing threat to public health, as they are becoming more common and harder to treat due to rising drug resistance. Among them, Aspergillus fumigatus has been classified as a critical pathogen by the World Health Organization (WHO). This filamentous fungus delivers spores in the air daily, which are constantly inhaled by humans. In people with weakened immunity, these spores can cause a range of lung diseases known as aspergillosis, with severity ranging from mild to life-threatening. Lung epithelial cells are the first cells of the respiratory tract to encounter inhaled spores. In a previous study, we showed that bronchial cells can prevent spore from developing into filaments, the invasive form of A. fumigatus that is responsible for tissue damage. This protective effect depends of on the recognition of a fungal protein called FleA. In the present study, we identified host cell proteins that bind to FleA and transport it into intracellular compartments. Our findings suggest that these proteins help bronchial epithelial cells to internalize fungal spores, thereby blocking their transformation into the invasive filamentous form.

BackgroundCytochalasans are a large family of fungal metabolites which inhibit actin polymerization and ultimately lead to a broad range of biological effects in different assays. Investigations into the biosynthesis of cytochalasans has revealed that the cytochrome P450 monooxygenase (P450s) tailoring enzymes possess a somewhat relaxed substrate-specificity and may accept structurally-related intermediates for oxidation, partly explaining the variety of structural variations observed in this family of molecules. In this study, we investigate a broad range of P450 enzymes via combinatorial biosynthesis to better understand their substrate scope and potential applications as biocatalysts. ResultsGenome mining enabled us to identify cryptic cytochalasan biosynthetic gene clusters (BGCs) in six different species of fungi, each with at least two P450 enzymes encoded. Comparative genomics identified a cryptic thioredoxin-like enzyme encoded in cytochalasan BGCs that co-occurs with the gene encoding a Baeyer-Villiger monooxygenase. Heterologous expression of seven P450s in Magnaporthe grisea mutant strains, lacking P450s required for pyrichalasin H biosynthesis, enabled functional characterization of three P450s, two of which were previously cryptic. The experimental results, combined with phylogenetic analysis of the P450 sequences, reveal subtle information regarding the structures of the associated cytochalasans and begins to explain why some P450s are inactive on the substrates available to them. ConclusionsThe P450 enzymes involved in cytochalasan biosynthesis are known to be site-selective in their native host but also possess intrinsic promiscuity due to being able to modify structurally-related analogues. By investigating a diverse set of P450s from characterized and cryptic BGCs, we were able to identify that the stereochemistry of functional groups around the cytochalasan backbone is more restrictive than the size of the macrocycle when introducing the P450 enzyme to non-native substrates.

BackgroundThe environment in which a fungus grows can directly influence their development, transmission, and pathogenic potential. This environment encompasses factors like nutrient availability, biotic and abiotic stressors, as well as host-derived chemical cues. In fungal pathogens, where conidia act as the infectious agents, the environment impacts the quantity and quality of these spores, thereby aOecting their ability to infect and kill hosts. In the present study, we investigated the effect of host-derived medium types on various phenotypes, including spore production, growth rate, and virulence in two entomopathogenic fungi, Metarhizium acridum and Metarhizium brunneum. Three medium types derived from insect material were compared to a standard laboratory medium. ResultsConidia produced on the insect-derived media exhibited enhanced sporulation and reduced time to sporulation, while conidial germination and maximum growth rate were comparable across medium types, suggesting that some of the medium-induced phenotypic effects were transient. Notably, conidia derived from two of the insect medium types demonstrated higher virulence, indicating that host-derived cues may prime virulence. ConclusionThese results highlight that the composition of growth substrates can regulate fungal reproductive strategies and virulence, with implications for developing high-throughput phenotyping and for the biotechnological optimization of mass production and efficacy of entomopathogenic fungi in biological control applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/711814v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@189013eorg.highwire.dtl.DTLVardef@1b0cedborg.highwire.dtl.DTLVardef@dccb4eorg.highwire.dtl.DTLVardef@1a77895_HPS_FORMAT_FIGEXP M_FIG C_FIG

Filamentous fungi have complex, three-dimensional growth patterns and a non-adherent nature, which can present challenges for live-cell imaging for quantitative assessment of dynamic cellular processes. To address these challenges, a live-cell imaging system has been modified to constrain the model fungus Aspergillus nidulans to growth in a single focal plane. This enables high-resolution time-lapse imaging of actin dynamics throughout development using a Lifeact actin marker. This system was used to perform kymographic analysis to quantify actin velocity and hyphal extension rates during early hyphal development. Results show two distinct growth phases: germ tube extension (0.58 m/min) and hyphal extension (1.52 m/min). Actin exhibited bi-directional transport along hyphae with biased movement toward the spore body. Actin was also observed re-localizing from hyphal tips to sites of septum formation indicating active redistribution of cytoskeletal resources based on cellular demands. This technological advancement overcomes longstanding limitations in fungal live-cell imaging and provides a new platform for quantitative systems-level analysis of mycelial development, offering new insights into the spatiotemporal coordination of cytoskeletal dynamics during filamentous growth.

The invasive fungal pathogen Pseudogymnoascus destructans is responsible for the collapse of several North American bat species through an infectious fungal skin disease known as White-Nose Syndrome (WNS). Recent transcriptomic studies have suggested that trace copper ion acquisition is essential for P. destructans propagation on its animal hosts. However, little is known about the mechanistic details of P. destructans adaptation occurring at the protein level. In this study, we report the global proteomic adaptation of P. destructans under chronic Cu-stress growth conditions employing chemically defined media. We identify 4340 P. destructans proteins, or approximately 47.8% of the predicted proteome, spanning a dynamic intensity range of six orders of magnitude. Chronic Cu-withholding stress leads to substantial alterations in the proteome, with 1398 differentially abundant proteins (DAPs) exhibiting statistically significant (p < 0.05) changes in protein levels compared to control growth conditions. We find that Cu-withholding stress induces increased levels of proteins associated with high-affinity Cu-acquisition, changes in intracellular superoxide dismutase (SOD) levels, and alterations in mitochondrial proteins related to aerobic respiration. In contrast, chronic Cu-overload stress leads to 390 DAPs (p < 0.05), which are more widely distributed across the proteome, with several DAPs associated with genomic stability and basic metabolism. Additionally, in this report, we present assessment of antisera products against intracellular and cell-surface protein targets of P. destructans that are effective for indicating Cu-withholding stress by western blotting.

European ash dieback caused by the invasive ascomycete species Hymenoscyphus fraxineus poses the most prominent danger to common ash trees (Fraxinus excelsior) in Europe. The disease is widely distributed in Europe and currently no efficient management strategy is available. Host-induced gene silencing and exogenous dsRNA applications have shown great potential for controlling fungal diseases in crop plants. In this study, we reported in silico evidence for the presence of a functional RNA interference pathway in Hymenoscyphus fraxineus. Moreover, we showed that the transgenic expression of a double stranded RNA (dsRNA) leads to inhibition of translation of its target polyketide synthase-like gene, a fungal endogene. We explored whether the dsRNA could be introduced exogenously and demonstrated that H. fraxineus can take up externally applied dsRNA molecules. This study highlights the RNA interference mechanism in H. fraxineus and suggests exoRNA applications as a promising approach to control European ash dieback.

AbstractEmergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterisation of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of H. capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences, to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall (1,3)-glucan by targeting the transcripts of the -glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus. ImportanceEmergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognised in recent years and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (1) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (2) methods for knocking down gene expression in E. africanus, and (3) the permanent disruption of genes through CRISPR/Cas9 gene editing.

The mitochondrion is a versatile organelle involved in diverse processes, such as cell death, metal homeostasis, plasma membrane and cell wall integrity, stress response, oxygen concentration, temperature, and metabolic adaptation, in addition to its role in generating energy. Consequently, mitochondrial fitness is essential for the pathogenicity of various organisms, including fungi. Cryptococcus neoformans is a fungal pathogen responsible for over 180,000 HIV-related deaths each year. In this study, we analyzed C. neoformans metabolic plasticity when grown with non-fermentable carbon sources and their impact on virulence and mitochondrial homeostasis. Growth on non-fermentable carbon sources increased thermotolerance, glucuronoxylomannan (GMX) content in the capsule, melanization rate, urease activity, biofilm formation, and virulence. Moreover, cells grown on non-fermentable carbon sources manifested increased mitochondrial number and activity. Conversely, mutants of the master regulator of mitochondrial biogenesis, the Hap complex, the catalytic subunit 1 of protein kinase A, or media supplementation with antioxidants, decreased the use of alternative carbon sources, capsule formation, melanin synthesis, urease activity, mitochondrial number, and resistance to both fluconazole and macrophage killing. Our results implicate mitochondrial homeostasis in virulence regulation via the PKA pathway, suggesting that targeting fungal mitochondrial homeostasis could be a therapeutic approach for cryptococcosis.

Peanut smut, caused by the fungus Thecaphora frezzii, is a significant disease of peanuts in Argentina. Infected plants have seeds replaced by a mass of dark teliospores, reducing yield and seed quality. To prevent the spread of the pathogen, several countries have limited import of raw peanuts from Argentina, a major grower and exporter. Following successful in vitro culture of the fungus in its haploid stage, we produced a chromosome-level genome assembly of the species for the first time. We compare this genome with those of 49 other species of true smut fungi, or Ustilaginomycetes, including species of medical, agricultural, and industrial importance, some of which are known as pathogens and others only as saprotrophic yeasts. At almost 39 Mb, T. frezzii has the largest genome of the smut fungi sequenced to date and the highest repetitive content. While it shares some core effectors with species of the distantly related and better studied Ustilago and related fungi, predicted effectors only found in T. frezzii or in Thecaphora suggest unique infection strategies. Comparisons among the 50 smut genomes also show that the 14 smut fungi observed only as yeasts share genomic traits such as low repeat content and generally smaller genomes. This supports the hypothesis that some smut fungi are adapted to saprotrophic growth as yeasts. The high-quality, annotated genome for T. frezzii will be a valuable resource for investigating the population dynamics and evolution of an economically important pathogen, as well as illuminating an understudied clade of smut fungi. Article summaryPeanut smut is a destructive and costly disease of peanuts in Argentina. For the first time, a high quality, annotated genome is presented for the causal agent, Thecaphora frezzii. This fungus has the largest and most repetitive genome of the true smut fungi, thus prompting comparison with 49 other species of smut fungi with available genomes, including non-pathogenic ones. While it shares some likely pathogenicity factors with well-studied smuts, it has many unique genes, a trait reflective of its evolutionary distance and likely novel mechanism for infection. This important genomic resource will benefit research regarding the evolution and adaption of T. frezzii, the development of diagnostic tools that enable rapid detection of it, and the study of smut fungi broadly.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

The objective of this study was to re-analyse the molecular phylogeny and/or the morphology of all species, which have been attributed to the so-far mono-generic fungal family Ambisporaceae. The genus Ambispora has been well-known for its spore bi-morphy described even from single spore clusters. Triple-walled spores are differentiated on sporiferous saccules, while mono-walled spores are formed on simple subtending hyphae. New phylogenetic analyses reveal dissimilarities of [&ge;]10% in partial nrDNA gene of three different stable phylogenetic clades and thus suggest the division of Ambispora into three genera, which simultaneously request for advanced morphological separations. These advances are primarily based on the more diverse spore wall composition of the ambisporoid-acaulosporoid morph rather than on the rather simple-glomoid morph. While all known species of the triple-walled morph have an evanescent to semi-permanent outer spore wall, i) Am. fennica, Am. brasiliensis, Am. gerdemannii and Am. nicolsonii have a smooth, permanent central spore wall (Am. fennica clade, A), ii) the central wall of Am. appendicula, Am. callosa, Am. leptoticha and Am. jimgerdemannii is alveolate (Am. appendicula clade, B), and iii) the central wall of Am. granatensis is smooth, but easily degraded, thus rather short-lived and not permanent but evanescent (Am. granatensis clade, C). In conclusion, species of the Am. fennica clade represent the genus Ambispora, while species of the Am. appendicula clade represent the new genus Appendiculaspora, and the mono-specific Am. granatensis clade represents the new genus Ephemerapareta. Species of an additional morph, with triple-walled spores, but apparently formed on subtending hyphae, and having a diagnostic reticulate, football-like middle wall, are here separated from the revised genus Ambispora based solely on morphological analyses, since molecular identification analyses so far failed and remained merely unknown. This later morph and genus is based on the type species Pelotaspora reticulata comb. nov, and on P. austrolatina sp. nov. Concomitant molecular phylogenetic and morphological analyses are needed to attribute not only Pelotaspora spp., but also those species, for which hitherto only the ambisporoid-glomoid morph has been observed correctly within the family Ambisporaceae. Without molecular analyses, such species with glomoid but unknown ambisporoid-acaulosporoid morph have to be retained within Ambispora.

The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in S. cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter and secreted phosphatase activity. These results indicate that both transcription factors display partially overlapping functions, responding differently to diverse stimuli, and that together they constitute a key component in the adaptation to a variety of stresses. Therefore, K. phaffii is an exceptional example among fungi that encodes two Pho4 functional transcription factors.

The SWI/SNF complex comprising of the catalytic subunit, Snf2, is a key regulator of gene expression and DNA damage repair in eukaryotic cell. Candida albicans Snf2 is known to regulate hyphal formation. In this paper, we have investigated the role of this protein in DNA damage response. We show that CaSnf2 is required for cell division as deletion of both copies of SNF2 leads to increased duplication time. The mutant cells form clumps with increased chitin and {beta}-glucan deposition on the cell wall. The altered cell wall phenotype leads to reduced uptake of genotoxic stressors leading to increased resistance to both methyl methane sulfonate (MMS) and hydroxyurea (HU). In addition, resistance of Casnf2{Delta} cells to MMS also appears to be mediated by upregulation of CaRAD9 expression by CaFun30, an ATP-dependent chromatin remodeling protein, and CaRtt109, a fungal-specific histone acetyltransferase. The response of Casnf2{Delta} to genotoxic stressors is at variance with the response of Scsnf2{Delta} mutant, highlighting the differences in DNA damage response/repair pathway between the two organisms. Finally, we show that Casnf2{Delta} mutants are extremely sensitive to azoles due to downregulation of multi-drug resistance pumps leading to reduced efflux of the drug.

Currently, the fungal class Archaeosporomycetes consists of one order, Archaeosporales with four families: Archaeosporaceae, Ambisporaceae, Geosiphonaceae, and Polonosporaceae. In the present study, the objective was to re-analyze the phylogeny and morphology of the Archaeosporomycetes from order to genus level. The different ecological strategies and, consequently, distinct evolutionary patterns of these taxa, as well as their morphological characters and other data updated here, suggest the need to divide Archaeosporales into four orders: (i) the type order Archaeosporales, (ii) Ambisporales ord. nov., both with four genera, (iii) Geosiphonales and (iv) Polonosporales ord. nov., both with single families and genera. Remarkably, the order Geosiphonales was described in the past, but was not considered in the Archaeosporomycetes until now. Phylogenetically, the four main clades (orders here proposed) of Archaeosporomycetes are well supported, with bootstrap values higher than 95% in all analyses, except Ambisporales/Ambisporaceae for RAxML-NG FBP analysis in the SSU tree (75%). Ecologically, this class includes three orders of arbuscular mycorrhizal fungi (AMF) forming symbiotic associations with plants, while Geosiphonales form an endocytobiosis with the cyanobacterium Nostoc. Morphologically, there are at least two AMF orders with spore bimorphism, which has not (yet) been described for Polonosporales. The only known species of Polonosporales, Polonospora polonica, forms spores directly on the neck of sporiferous saccules and the spores can morphologically be differentiated from all other taxa in Archaeosporomycetes by the formation of three permanent, rather thick spore walls, of which two form de novo during spore formation. The outer spore wall of Archaeosporales and Ambisporales are semi-permanent, evanescent or even short-lived, or show multiple fissures during aging, when it is more resistant. Ambisporales can easily be differentiated from Archaeosporales for instance by larger spores of the acaulosporoid morph and thicker spore walls. Our phylogenetic analyses suggested that Archaeosporales can be divided into two families: Antiquisporaceae that was described to form intraradical hyphae, vesicles and spores, staining darkly in Trypan blue, and Archaeosporaceae whose hyphae generally do not or only faintly stain in this reagent, and vesicles and intraradical spores have been rarely, if ever reported.

Glutathione has important roles in diverse infections, yet its involvement in the interaction between the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd) and its amphibian hosts is still unclear. Using in vitro assays and a cell infection model, we examined how glutathione influences Bd virulence traits and cellular host disease resistance. For Bd, inhibition of glutathione reductase rapidly killed zoospores, indicating that glutathione is essential for this pathogen. In addition, exposure to exogenous glutathione promoted the potential for virulence through accelerated and increased zoospore release. In host amphibian cells, Bd infection decreased intracellular glutathione content and increased reactive oxygen species, suggesting that chytridiomycosis pathogenesis may involve oxidative stress. Depletion of host glutathione before exposure to Bd increased infection severity and Bd growth, whereas amphibian cells with slightly elevated glutathione levels were partially protected against Bd. However, manipulation of host glutathione levels after the establishment of Bd infection did not impact its intracellular growth, implying that the host glutathione-mediated resistance only occurs during the initial Bd invasion process. Importantly, this effect of glutathione on host resistance is not a general response to pathogens, as it was not observed in cells exposed to viral pathogen FV3. As glutathione increased both infectious zoospore production and host resistance to zoospore infection, our study suggests that this antioxidant may play an important role in the host/pathogen interaction during chytridiomycosis. Thus, environmental conditions and therapeutic approaches that affect glutathione systems in the host and/or pathogen have the potential to alter chytridiomycosis dynamics and should be further explored.

Drug interaction outcomes-synergism, additivity, or antagonism-represent complex phenotypes. While drug repurposing aims to identify compounds that potentiate conventional antifungals, when given in combination, the modulators of these interactions in fungi remain largely unexplored. We hypothesize that the response to repurposed-conventional antifungal pairs is a complex trait modulated by genetic and environmental factors. To study the impact of genotype on the outcome, we screened six diverse Saccharomyces cerevisiae isolates, including clinical, wild, and fermentation strains, for their responses to combinations of ibuprofen with either clotrimazole or caspofungin. We evaluated the role of the environment using rich and minimal media and assessed the influence of assay type by comparing solid- and liquid-rich media assays. Our results reveal that ibuprofen-clotrimazole interactions are highly dynamic, predominantly antagonistic, with limited synergy observed. These outcomes are significantly modulated by genetic background, media composition, assay type, and, in specific genotypes, even by the drug dosage, reflecting a complex, multi-parametric phenotype. However, the ibuprofen-caspofungin combination is more predictable, exhibiting only synergy or additivity. Interaction outcomes correlate with baseline sensitivity to caspofungin: caspofungin-resistant isolates consistently demonstrate synergy, while sensitive strains exhibit additivity. These findings shift the paradigm of drug discovery by demonstrating that synergism and antagonism are not static properties of drug pairs but are dynamic, context-dependent outcomes. This study highlights the need to use clinically relevant models and patient-specific isolates before clinical application, as drug interactions cannot be generalized from a single dosage, strain, or environmental condition.

BackgroundTalaromycosis, caused by the fungus Talaromyces marneffei, is a leading cause of HIV-associated death in Southeast Asia. Current culture-based diagnosis only identifies late-stage infection, limiting understanding of disease burden and disease spectrum. We evaluated the clinical performance of anti-Mp1p IgM and IgG enzyme immunoassays (EIA) for talaromycosis diagnosis. MethodsThis diagnostic study included 423 adults with advanced HIV disease and culture-confirmed talaromycosis as cases, and 206 non-talaromycosis individuals with and without HIV who have never traveled to Asia as controls. Anti-Mp1p IgM and IgG antibodies were measured using conventional double-sandwich EIA. Diagnostic performance was assessed using the healthy control group and the HIV control group separately. Assay cut-offs were based on both the Youden index generated from the receiver operating characteristic curves, and separately using a pre-defined specificity of 95%. ResultsAt the pre-defined 95% specificity, IgM had low to moderate accuracy of 62.3% and 87.9%, and a low sensitivity of 8.3% and 21.3%, when evaluated with healthy and HIV controls, respectively. IgG had similarly low accuracy of 52.2% and 78.4%, and a low sensitivity of 21.5% and 30.5%, when evaluated using healthy and HIV controls, respectively. Both IgM and IgG assays performed better in HIV controls than healthy controls. ConclusionsThe anti-Mp1p IgM and IgG EIAs show low utility for the diagnosis of acute talaromycosis. However, at the high specificity cut-off of 95%, the assays have utility in the detection of T. marneffei exposure at both individual and population levels, and. provides a foundation for future sero-epidemiological studies. IMPORTANCETalaromycosis, caused by the dimorphic fungus Talaromycosis marneffei endemic in Southeast Asia, southern China, and northeastern India, is an invasive fungal infection that causes over 25,000 cases and 6,000 deaths annually but remains neglected in the global health community. Current diagnosis requiring culture-based testing is too slow, often resulting in patient death before treatment can begin. This study presents the first large-scale clinical evaluation of antibody tests for talaromycosis. While the antibody tests showed limited sensitivity for diagnosing acute disease, the high specificity makes them useful in determining prior exposure to T. marneffei, providing a new tool for public health investigation of disease prevalence at a population level, and for clinicians to identify individuals at risk for disease reactivation who may benefit from prevention strategies. The research provides important groundwork for improving disease control efforts in regions where this neglected infection is endemic.

Filamentous fungi, such as Aspergillus species, use microtubule transport to move early endosomes. Other cargos, such as peroxisomes and mRNAs, "hitchhike" on early endosomes to move throughout the long hyphae of these organisms. In Aspergillus nidulans, peroxisomes hitchhike on early endosomes using the endosomal protein PxdA and the peroxisomal protein AcbdA. The HookA adaptor protein links endosomes to microtubule motors. Here, we set out to explore the physiological functions of peroxisome hitchhiking and endosome motility. A. nidulans has a complex life cycle that includes asexual and sexual reproduction. A. nidulans and other fungi within the Pezizomycotina subphylum are also notable for the vast number of secondary metabolites they produce. Light and other environmental conditions influence developmental decisions and secondary metabolite production. Here, we found that sexual reproduction is favored in the absence of endosome motility, even in the light, which normally promotes asexual reproduction. RNA sequencing of strains lacking endosome motility showed altered expression of genes involved in development. Unexpectedly, we observed altered expression of genes involved in secondary metabolism in strains lacking endosome motility and peroxisome hitchhiking. Using mass spectrometry, we found that the loss of endosome motility affected the biosynthesis of secondary metabolites, including sterigmatocystin, a carcinogenic mycotoxin that is a food contaminant. Finally, in a pathogenic species, Aspergillus fumigatus, we found that deletion of its PxdA homolog also significantly altered secondary metabolite production. Our work uncovers an unexpected link between organelle motility, developmental decisions in response to light, and secondary metabolite production in filamentous fungi.