Journal of Fungi

Antifungal susceptibility testing quantifies end-point fungal biomass in liquid cultures initiated from non-invasive yeast or spore morphologies. However, end-point analyses obscure informative spatio-temporal responses to drug exposures. In the major fungal pathogens Aspergillus fumigatus and Candida albicans we used microfluidic-coupled, fluorescence-mediated live-cell imaging to capture the real-time responses of fungal hyphae to clinical concentrations of AmBisome or Caspofungin. In both fungi, AmBisome exposure caused rapid growth arrest, extensive hyphal vacuolation and membrane blebbing. Responses to Caspofungin exposure were slower with initial lytic effects occurring after 1.5 or 4 hours in A.fumigatus and C.albicans, respectively. Whilst C.albicans hyphae undergo unsalvageable hyphal lysis in response to Caspofungin, A.fumigatus exhibit several compensatory growth behaviours, including a novel resuscitative growth form, that circumvent lytic events to maintain apical and sub-apical hyphal growth. This study reveals how the differing biologies of the two pathogens affected outcomes and contributes to the highly disparate rates of antifungal efficacy amongst commonly used drugs, where spore/yeast-derived inhibitory doses may underestimate the dose required to arrest/kill the invasive hyphal morphotypes of fungal pathogens in vitro.

Under certain conditions, fungi can rapidly change the size of their cells. For example, it is known that the cells of many yeast species under hyperosmosis instantly and reversibly shrink entirely, without plasmolysis, with a decrease in volume of up to 70%. There is evidence that filamentous fungi can also instantly change the diameter of their unspecialized hyphae. This property is fundamental but requires detailed study. In this large-scale study (involving more than 50,000 cells measured) using light microscopy, the ability of three unrelated basidiomycete species to rapidly change the diameter of their hyphae under various factors was analyzed. It was found that all three fungi respond similarly to moderate hyperosmotic shock or detergent treatment, shrinking by an average of 12-14% in diameter. However, inhibitors of actin assembly can cause either expansion or shrinkage of hyphae or have no effect on a fungus. These results, along with previously established features of the macroinvagination systems of the plasma membrane in basidiomycetes, are important for understanding the complex structural-protective physiological mechanisms responsible for the survival and continuous functioning of fungal cells in unstable environmental conditions.

The response of filamentous fungi towards recalcitrant carbohydrates is majorly governed by transcriptional activators of cellulase genes; however, little is known about the downstream events beyond transcription. We show here in Penicillium funiculosum that increasing the expression of a transcriptional activator CLR-2 in the catabolically derepressed strain, {Delta}Mig1, didnt exhibit a synergistic effect on cellulase production unless Ca2+ was simultaneously increased. The RNA-seq screen for Ca2+-activated kinases identified SNF1-AMPK and SSP1-AMPKK as being specific to cellulose induction. Deletion of snf1 led to negligible secretion of cellulase upon induction. Quantitative whole-cell proteomics followed by chemical-genetic experiments with snf1-deleted strain showed that Ca2+-signaling channelizes carbon, nitrogen and energy sources towards cellulase production. Further, Ca2+-signaling phosphorylates SNF1-AMPK via SSP1, which in turn downregulates the phospho-HOG1 levels, leading to stimulus for cellulase secretion during carbon stress. The findings reported here are significant for understanding fungal pathology and developing second-generation biorefineries.

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.

Fungi possess several transcription factors with a characteristic velvet domain for DNA-binding and homo- or heterodimerization, which is structurally similar to the mammalian NF-B Rel homology domain. Velvet dimers control fungal development, virulence and mycotoxin formation. VelB is the only regulator, which carries an intrinsically disordered domain (IDD) within the velvet domain. The IDD as well as the positioning within VelB is conserved in the fungal kingdom. Intrinsically disordered regions contribute to transcription activation and DNA binding and frequently appear in eukaryotic transcription factors. The VelB IDD provides selective heterodimerization as well as protein stability control. The IDD is not required for the formation of the VelB-VeA heterodimer of Aspergillus nidulans or Verticillium dahliae, but promotes the formation of the VelB-VosA heterodimer. The IDD destabilizes VelB single molecules and also balances its distribution and ratio between both velvet heterodimers. These balances contribute to control appropriate mycotoxin production and sexual development. Herewith, the VelB IDD represents a novel control mechanism of velvet protein stability and heterodimer formation for precise priming of fungal development.

The fungal phytopathogen Zymoseptoria tritici, causal agent of the economically damaging Septoria tritici blotch of wheat, is different from most foliar fungal pathogens in that its germination occurs slowly and apparently randomly after arrival on the leaf surface and is followed by a potentially prolonged period of epiphytic growth and even reproduction, during which no feeding structures are formed by the fungus. Thus, understanding the cues for germination and the mechanisms that underpin survival in low-nutrient environments could provide key new avenues for disease control. In this work, we examine survival, culturability, and virulence of spores following transfer from a high nutrient environment to water. We find that a sub-population of Z. tritici spores can survive and remain virulent for at least 7 weeks in water alone, during which time multicellular structures split to single cells. The fungus relies heavily on stored lipids; however, if cell suspensions in water are dried, the cells survive without lipid utilisation. Changes in gene expression in the first hours after suspension in water reflect adaptation to stress, while longer term starvation (7 days) induces changes particularly in primary metabolism and cytochrome P450 (CYP) gene expression. Importantly, we also found that Z. tritici spores are equally or better able to survive in soil as in water, and that rain-splash occurring 49 days after soil inoculation can transfer cells to wheat seedlings growing in inoculated soil and cause Septoria leaf blotch disease.

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. Deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for expression of the conidiation regulatory genes brlA, abaA and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both the neutropenic and corticosteroid-immunosuppressed mouse models.

Aspergillus flavus colonizes oil-seed crops contaminating them with aflatoxins, highly carcinogenic mycotoxins that cause severe health and economic losses. Genetic studies may reveal new targets for effective control strategies. Here we characterized a putative WOPR transcription factor gene, osaA, in A. flavus. Our results revealed that osaA regulates conidiation and sclerotial formation. Importantly, deletion of osaA reduces aflatoxin B1 production, while, unexpectedly, transcriptome analysis indicated upregulation of aflatoxin biosynthetic genes, suggesting post-transcriptional or cofactor-mediated regulation. Cyclopiazonic acid production also decreased in absence of osaA. In addition, the osaA mutant exhibited upregulation of genes in the imizoquin and aspirochlorine clusters. Moreover, osaA is indispensable for normal seed colonization; deletion of osaA significantly reduced fungal burden in corn kernels. Aflatoxin content in seeds also decreased in the absence of osaA. Furthermore, deletion of osaA caused a reduction in cell-wall chitin content, as well as alterations in oxidative stress sensitivity, which could in part contribute to the observed reduction in pathogenicity. Additionally, promoter analysis of osaA-dependent genes indicated potential interactions with stress-responsive regulators, indicated by an enrichment in Sko1 and Cst6 binding motifs. Understanding the osaA regulatory scope provides insight into fungal biology and identifies potential targets for controlling aflatoxin contamination and pathogenicity. Key ContributionAspergillus flavus osaA controls morphological and chemical development, as well as phytopathogenicity, and could be a promising target for a control strategy against A. flavus to reduce health risks and economic losses associated with aflatoxin contamination.

Progresses in geothermal energy and deep drilling technologies have opened a new window into the terrestrial subsurface. This provides direct access to deep geothermal fluids used to produce heat and electricity, creating an opportunity to isolate and characterize novel microbial strains from these extreme habitats. In this study, we report the co-isolation of two fungal strains. Penicillium citrinum (strain HEK1) was isolated first and thought to be axenic. However, upon exposure to stress (frost and ethanol), a second strain, corresponding to the dimorphic yeast Meyerozyma guilliermondii (strain HEK2), appeared in HEK1 cultures. Strain HEK2 appeared first in the cultures and was followed by the subsequent re-growth of strain HEK1, underscoring their close relationship. Moreover, strain HEK2, able to switch from yeast cells to pseudohyphae when growing alone, did not produce pseudohyphae when in direct contact with strain HEK1. Altogether, our results indicate an intricate interaction between these strains that may allow them to thrive in the deep subsurface. These two fungi represent the first fungal strains isolated from deep geothermal fluids. Their presence within the fluids was confirmed through molecular analysis. The isolation of these strains emphasizes the importance of considering fungi when investigating microbial diversity in subsurface geothermal environments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/599737v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@fdf699org.highwire.dtl.DTLVardef@148b751org.highwire.dtl.DTLVardef@fb37e9org.highwire.dtl.DTLVardef@113b911_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst fungal strains isolated from a geothermal power plant C_LIO_LIThe two fungal strains were co-isolated from a geothermal fluid used for heat production C_LIO_LISurprising isolation of the cell-switching yeast upon stress exposure of an apparently axenic culture of the filamentous fungus C_LIO_LIFungal strains with high resistance to stressors and no apparent competition for carbon sources C_LI

Botrytis pseudocinerea is a cryptic fungal species, morphologically indistinguishable from the notorious plant pathogen Botrytis cinerea, yet their distinct ecological behaviours suggest fundamental biological differences. This study resolves the paradox of why B. pseudocinerea, despite intrinsic resistance to the fungicide fenhexamid, often fails to dominate agricultural ecosystems. Remarkably, we demonstrate that B. pseudocinerea VD165 exhibits superior vegetative growth and stress tolerance compared to B. cinerea B05.10, coupled with heightened virulence on solanaceous hosts like tomato and tobacco. A comprehensive bio-guided chemical investigation combined with targeted gene expression analysis reveals a fundamental schism in its secondary metabolism. The VD165 isolate of B. pseudocinerea produces a potent phytotoxic cocktail of botcinin polyketides, a strategy supported by the constitutive and infection-induced strong upregulation of the Bcboa6 and Bcboa9 biosynthetic genes. Critically, we establish that it has completely lost the botrydial sesquiterpene pathway, a primary virulence factor in B. cinerea. The significant accumulation of the upstream terpene precursor mevalonolactone provides definitive biochemical evidence for this truncated pathway. This metabolic switch--the evolutionary loss of one major toxin gene cluster and the compensatory upregulation of another--mirrors the co-regulatory mechanism previously demonstrated through genetic knockout of the botrydial pathway in B. cinerea and is a pivotal event that has shaped the unique pathogenic identity of B. pseudocinerea. This finding provides a model for how loss-of-function events in secondary metabolism can redefine host specificity and virulence in fungal pathogens--an evolutionary principle applicable across microbial taxa. Author SummaryIn our study, we investigated a biological puzzle. We focused on Botrytis pseudocinerea, a "cryptic" fungus that looks identical to the common grey mould pathogen, Botrytis cinerea. This hidden species is resistant to a major fungicide, yet paradoxically, it often fails to outcompete its fungicide-susceptible sibling in treated agricultural fields. We wanted to know why. We discovered that B. pseudocinerea has undergone a major evolutionary "trade-off". During its evolution, it completely lost the gene cluster for botrydial, a primary toxin used by B. cinerea to infect plants like grapes. To compensate, its genome has permanently "super-charged" the production of a different chemical cocktail--a potent family of toxins called botcinins. This metabolic switch defines its identity: it is less effective on hosts like grapes, but has become a hyper-virulent specialist on other hosts, like tomato and tobacco. Our work provides a clear model of how "evolution by subtraction"--losing a key function--can be a powerful force in creating a new, specialized pathogen, solving an ecological mystery in the process.

C. neoformans is notorious for causing severe pulmonary and central nervous system infections, particularly in immunocompromised patients. High mortality rates, associated with its tropism and adaptation to the brain microenvironment and its drug resistance profile, makes this pathogen a public health threat and a World Health Organization (WHO) priority. In this study, we reconstructed GSMM iRV890 for C. neoformans var. grubii, providing a promising platform for the comprehensive understanding of the unique metabolic features of C. neoformans, and subsequently shedding light on its complex tropism for the brain microenvironment and potentially informing the discovery of new drug targets. The GSMM iRV890 model is openly available in the SBML format, and underwent validation using experimental data for nitrogen and carbon assimilation, as well as specific growth and glucose consumption rates. Based on the comparison with GSMMs available for other pathogenic yeasts, unique metabolic features were predicted for C. neoformans, including key pathways shaping the dynamics between C. neoformans and the human host, and underlying its adaptation to the brain environment. Finally, predicted essential genes from the validated model are explored herein as potential novel antifungal drug targets.

An ability of fungi to undergo hyphal constriction is important for fungal ecology and diseases. In the rice blast fungus, Magnaporthe oryzae, hyphal constriction is required to traverse host cell junctions through pit fields. However, little is known about genetic underpinnings of hyphal constriction, except the requirement of a mitogen-activated protein kinase, Pmk1. Here we demonstrate that a simple in vitro assay based on nitrocellulose membrane allows investigation of the genetic basis for hyphal constriction. Using the assay, we found that the constriction limit of M. oryzae hypha lies between 0.22 and 0.3 m, and that a histone modification might be involved in hyphal constriction. RNA-seq experiments combining our assay and {Delta}pmk1 showed that hyphal constriction relies on transcriptional changes of genes implicated primarily in membrane and cell wall-related cellular processes in Pmk1-dependent and/or Pmk1-independent manner. Furthermore, our assays with diverse fungal species suggest correlation between hyphal constriction and fungal lifestyles. Our study reveals that hyphal constriction can be induced without host-derived cues and provides molecular and evolutionary insights into a fundamental process to infection of host plant.

Mucormycosis, a rare but fatal invasive fungal infection, affects immunocompromised patients. Despite recent advancements in diagnostic tools and treatments, mortality rates remain high, reaching 79% at ninety days in disseminated forms. Quantitative Polymerase Chain Reaction (qPCR) based methods are now widely available, and help to discern colonisation and infection, although this remains a real challenge. This study aims to describe the epidemiological and clinical characteristics of a French cohort of patients with presence of Mucorales in clinical samples. With a retrospective monocentric study from 2017 to 2022, we investigated risk factors associated with proven or probable mucormycosis and in-hospital mortality. Patients included had at least one Mucorale-positive culture, or positive PCR assay detecting Mucorales. Clinical, microbiological, and hospital management data were collected and analyzed using univariate and multivariable statistical models. Among our 85 identified patients, in-hospital mortality was significantly associated with positive cultures obtained from deep respiratory samples (OR=16.6, p=0.017) and diagnosis based on EORTC+PCR criteria (OR=12.4, p=0.016). Principal-component analysis revealed a homogeneous group of patients with positive deep respiratory swabs, haemopathy, ICU admission and a greater death ratio. We completed with an expert analyses on diagnosis, which revealed diagnostic variability with an overall Krippendorffs alpha about 0.32. Mucormycosis remains a challenging condition due to its rarity, severity, and diagnostic limitations. Incorporating PCR into diagnostic criteria and implementing early and tailored management for at-risk patients may improve clinical outcomes. Prospective studies are required to validate these findings and refine management strategies for this life-threatening infection.

IntroductionParasites can modify host behavior to ensure their own growth and transmission. Multiple species of the fungi Ophiocordyceps infect ants, but in a species-specific manner; one fungal species co-evolved to successfully modify the behavior of one ant species. However, several characteristics of the behavioral modification seem to be similar across different Ophiocordyceps-ant systems, including a preference for the time of the day for manipulating host behavior. In this study, we explored the various mechanisms via which the circadian clock of Ophiocordyceps might be playing a role in modifying host behavior. We studied O. camponoti-floridani that modifies the behavior of its ant host Camponotus floridanus. To separate the role of the clock in behavior manipulation, from its role in growth and survival, we compared the daily gene expression profile of O. camponoti-floridani to a generalist, non-manipulating fungal parasite, Beauveria bassiana, which also successfully infects the same ant host. ResultsMajority of the 24h rhythmic O. camponoti-floridani genes show peak expression before or at the transitions between light and dark. Rhythmic genes in O. camponoti-floridani, for which B. bassiana lacks an ortholog, were overrepresented for enterotoxin genes. Around half of all genes that show 24h rhythms in either O. camponoti-floridani or B. bassiana showed a consistent difference in their temporal pattern of daily expression. At the halfway mark in O. camponoti-floridani infections, when diseased ants show a loss of 24h rhythms in daily foraging, several fungal clock genes, including Frequency, showed differential expression. Network analyses revealed a single gene cluster, containing White Collar 1 and 2, that showed overrepresentation for genes oscillating every 24h in liquid culture as well as genes differentially expressed while growing inside the ant head. ConclusionThis study identifies several sets of putatively clock-controlled genes and biological processes in O. camponoti-floridani that likely plays a role in modifying the behavior of its ant host. Differential expression of O. camponoti-floridani clock genes or 24h-rhythmic genes during infection is suggestive of either a loss of daily rhythm or a change in the amplitude of rhythmic gene expression. Both possibilities would suggest that a disease-associated change occurs to the functioning of the O. camponoti-floridani clock, and its output, while the fungi grows inside the ant head.

Mucorales are basal fungi that opportunistically cause a fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic organisms. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis. Author SummaryThe knowledge regarding the role of epigenetic modification in regulating gene expression in early diverging fungi is scarce, despite they represent an important fraction of the fungal kingdom. The order Mucorales, which causes the lethal infection known as mucormycosis, is not an exception. There is an urgent need to enhance our understanding of the biology of these fungi to develop effective treatments for mucormycosis, which are currently absent due to the natural resistance of Mucorales to most antifungal drugs. This work represents the first investigation into the role of the methylation of lysine 4 on histone 3 (H3K4) in a mucoralean fungus. This was accomplished by the generation of deletion mutants in the set1 gene, which encodes the specific H3K4 methyltransferase. Phenotypic analyses of these mutants suggest that H3K4 methylation regulates physiology, development, cell wall integrity, and DNA repair. Furthermore, our findings indicate that it also contributes to the virulence of M. lusitanicus, as strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo.

Most Eukaryotic organisms produce nitric oxide (NO); however, the mechanisms underpinning NOs biosynthesis are only known in animals. In animals, there seems to be a non-described additional system for producing NO in many cell types, including blood vessels where NO is essential for blood pressure control. NO is known to be a signalling molecule of the innate immunity system in plants and fungi although no NO generation has yet been described. In the plant pathogenic fungus Fusarium graminearum, we demonstrate an extra NO-producing system in fungi that seems also present in mammals and plants and, thus, likely the evolutionary original. The discovered NO-producing enzymes are already well-known sterol-producing enzymes with more than one function. Both these enzymes are targets for statins and the major fungicides; thus, the NO production of the new system has consequences for agriculture (pathogen resistance and control) and medicine (blood pressure control, immunity and sepsis).

The atromentin synthetase gene cluster is responsible for catalyzing the precursor pigment atromentin, which is further catalyzed into hundreds of different pigments that span different taxa in the Basidiomycota and is a distinguished feature of Boletales. Previous work identified co-transcription of the two essential clustered atromentin genes (the atromentin synthetase (NPS) and the aminotransferase) by inducible pigment conditions and also conserved genetic elements in the promoter regions (motifs). For this work, we found that the NPS and its promoter motif appeared to follow the same evolutionary path as the mushrooms. The NPS appears to predate Boletales and originate in Agaricomycetes, and with convergent/parallel evolution that allowed ascomycetes to produce atromentin. Additionally, a consensus of the intron-exon gene structure for basidiomycetous, atromentin-catalyzing NPSs was identified whereby a significant deviation occurred in the paraphyletic group, Paxillaceae. This gene structure was not present in NPSs in Aspergilli. Lastly, we found a putative TATA box adjacent to the palindromic motif of NPS, indicating (co-)transcriptional control by a TATA(-like) binding transcription factor. Combined with previous decades worth of research, our results support that not only can atromentin derivatives be used for chemo-taxonomy, but also atromentins genetic basis. Future work using the putative promoter motif will provide new insight into which (co-)transcription factor may be responsible for the transcriptional control of atromentin synthetases.

The fungi kingdom is composed of eukaryotic heterotrophs, which are responsible for balancing the ecosystem and play a major role as decomposers. They also produce a vast diversity of secondary metabolites, which have antibiotic or pharmacological properties. However, our lack of knowledge of gene function in fungi precludes us from tailoring them to our needs and tapping into their metabolic diversity. To remedy this, we gathered genomic and gene expression data of 19 most widely-researched fungi to build a database, fungi.guru, which contains tools for cross-species identification of conserved pathways, functional gene modules, and gene families. We exemplify how our database can elucidate the molecular function, biological process and cellular component of genes involved in various biological processes, by identifying a secondary metabolite pathway producing gliotoxin in Aspergillus fumigatus, the catabolic pathway of cellulose in Coprinopsis cinerea and the conserved DNA replication pathway in Fusarium graminearum and Pyricularia oryzae. The database is available at www.fungi.guru.

ABSTRACTCandida haemulonii comprises a group of pathogenic fungi known for their resistance to primary antifungal treatments. Infections caused by these pathogens present substantial challenges due to the difficulties in accurate identification. Extracellular vesicles (EVs) released by these fungi play a critical role in the pathogen-host interaction, potentially influencing antifungal resistance and virulence. Previous research by our group indicates that EVs contain immunogenic particles capable of impacting the hosts immune response. Understanding the composition of these EVs is crucial for elucidating the mechanisms underlying resistance and virulence in C. haemulonii var. vulnera. This study aims to investigate the contents of EVs from C. haemulonii var. vulnera using proteomic and microRNA sequencing tools, providing insights into their role in adaptation, survival, and the progression of infections. Our findings reveal key proteins transported by EVs, including BMH1, TEF1, CDC19, and PDC11. These proteins are involved in various cellular processes, such as the alteration of cell wall structure, biofilm formation, and facilitation of morphological changes, among others. Additionally, we observed that miRNA-like molecules transported within EVs are linked to the electron transport chain and regulation of the citric acid cycle, which are metabolic processes associated with virulence factors and rapid adaptation to diverse hosts or environments. In this context, our findings provide a novel perspective on fungal EVs, highlighting their potential as targets for therapies. Therefore, these vesicles may reflect the expression levels of regulatory molecules crucial for the survival, pathogenicity, and virulence of C. haemulonii var. vulnera. IMPORTANCEThe study of Candida haemulonii complex holds substantial clinical significance due to its notable resistance to conventional antifungal therapies and the complex challenges inherent in its specific identification. This research focuses on cargo of EVs released by these fungi, which play an essential role in pathogen-host interactions, influencing fungal pathogenicity. EVs contain immunogenic particles that can modulate the hosts immune response. Proteomic and microRNA analyses of EVs from Candida haemulonii var. vulnera have identified key proteins and miRNAs involved in cellular processes such as metabolic adjustment, biofilm formation, and modulation of cytoplasmic functions. These components are essential for the adaptation, survival, and progression of infections. This study offers novel insights into fungal EVs, underscoring their potential as targets for therapeutic intervention. By elucidating the mechanisms underlying the rapid adaptation of Candida haemulonii, the research enhances our understanding of the pathogenicity of this emerging yeast.

Under low-nutrient conditions, Arthrobotrys oligospora and other NTFs can differentiate their mycelia into specialized trapping devices for capturing prey as their nutritional source. Using energy-dispersive X-ray spectroscopy (EDX) in conjunction with transmission electron microscopy (TEM), we identified that the characteristic electron-dense bodies in trapping devices contained more iron than vacuoles and mitochondria. Meanwhile, fungal mycelial cells used effective desferriferrichromes for iron chelation and storage. Complex bioassays showed that electron-dense bodies represent a novel type of microbial iron storage particle and trapping devices in A. oligospora function as an unprecedented phenotypic system for iron storage. Unexpectedly, all NTFs lack a crucial Ccc1-mediated vacuolar iron detoxification mechanism, which is conserved in most fungi. Inserting the Ccc1 gene cloned from yeast into A. oligospora significantly reduced formation of trapping devices and inhibited nematicidal activity. Notably, Bayesian relaxed molecular clock analysis indicated that the loss of Ccc1-mediated vacuolar iron storage occurred during the Late Paleozoic Ice Age, while the origin of the trapping devices and the acquisition of desferriferrichrome biosynthesis were strongly associated with significantly elevated temperatures. Temperature bioassays demonstrated that the formation of trapping devices is highly temperature-dependent, with free iron content in mycelial cells being inversely proportional to temperature, consistent with that A. oligospora is sensitive to high temperatures and fails to grow above 30{degrees}C. Our findings revealed that global temperature fluctuations are a crucial driver of the genetic evolution of NTFs, as a catalyst for the origin of trapping devices, which are a novel phenotypic indicator of eukaryotic iron overload. Author summaryWe found that a unique group of carnivorous fungi has evolved specialized trapping devices to sequester excess iron, compensating for the absence of the crucial Ccc1-mediated vacuolar iron detoxification pathway, which is conserved in most fungi. Furthermore, we report for the first time that elevated temperature is a non-trophic factor that induces iron overload in eukaryotes, as iron content and composition in fungal mycelia are negatively correlated with temperature. Our findings suggest that nematode-trapping fungi could serve as a potential eukaryotic model for investigating the dynamic regulation mechanisms of iron homeostasis, which could contribute to the development of therapies for iron overload-related diseases in humans. In humans, iron overload leads to tissue damage, particularly in the cardiovascular system. It has long been assumed that iron overload occurs when iron intake is increased over an extended period, either through repeated red blood cell transfusions or enhanced absorption from the gastrointestinal tract. Caucasians are particularly susceptible to iron overload and the complications of hemochromatosis due to a higher incidence of mutations in the homeostatic iron regulation gene within this population. The discovery of eukaryotes exhibiting an iron overload phenotype when exposed to heat holds significant implications for developing treatments and strategies for human iron overload disorders.