Journal of Fungi

Colletotrichum graminicola causes significant losses of the staple crop maize worldwide. The fungus produces two distinct asexual spore types, oval and falcate conidia, which show unique processes in development and plant interaction. Based on genome resequencing of our laboratory strain (CgM2/ M1.001), we investigated the gene expression profiles of oval and falcate conidia during development and early leaf infection using RNA-seq. Our results reveal specific gene expression profiles between the two spore types, indicating fundamental differences in their developmental programs that reflect different modes of infection. We identified expression patterns discriminating both conidia types from mycelium and spore type-specific ones for genes encoding transcription factors, conserved fungal developmental genes, transporters, genes of secondary metabolite clusters, and pathogenicity-related functions, including effectors and carbohydrate-active enzymes (CAZymes). Our study shows that despite the identical genomic basis, oval and falcate conidia show unique transcriptomes across vegetative development and early plant interaction. Taking together, these results provide new insights into the molecular mechanisms determining the biology of C. graminicola and its interaction with the plant host.

Mucormycosis is among the most lethal fungal infections in clinical medicine, with crude mortality rates between 50 and 90% that have improved little over decades of antifungal development. Alveolar macrophages constitute the primary cellular defense against inhaled Mucorales spores, yet quantitative comparative data on phagocytic outcomes across the taxonomic breadth of this fungal order are absent from the literature. We co-incubated 62 Mucorales strains drawn from nine phylogenetic groups and 12 countries with MH-S murine alveolar macrophages and with Dictyostelium discoideum amoeba, at multiplicities of infection (MOI) of 1 and 5 and at incubation times of 1.5 and 3 hours. Automated phagocytic index quantification was combined with spore morphometric measurements and geographic metadata. Macrophage phagocytic indices differed highly significantly among the nine phylogenetic groups across all experimental conditions (Kruskal-Wallis H = 394-780; p = 10^-163 to 10^-74). Group 1 (Absidia and Cunninghamella species) showed the lowest phagocytic index; Groups 5 (Rhizopus arrhizus) and 6 (Gilbertella persicaria and Mycotypha species) showed the highest. Larger spore area correlated negatively with macrophage phagocytic index. In the central comparative finding, Spearman rank correlations between macrophage and D. discoideum phagocytic indices were near zero across all four conditions (r = -0.07 to +0.11; all p > 0.8), demonstrating that protozoan phagocytosis does not predict the mammalian macrophage phagocytic hierarchy. Kinetic analysis further revealed, for the first time in any Mucorales or Mucormycotina species, evidence of vomocytosis in MH-S macrophages challenged with Group 8 (Mucor clade II) spores at MOI 1, and in D. discoideum across four groups at MOI 5 after 3 hours. These findings establish a quantitative, phylogenetically resolved framework for Mucorales innate immune recognition, demonstrate that the evolutionary training ground hypothesis does not hold at the order level for Mucorales, and identify vomocytosis as a previously unrecognised immune evasion mechanism in Mucormycotina.

Fungal mycoparasitism is commonly viewed as a broadly conserved lifestyle relying on the secretion of cell wall-degrading enzymes and specialized metabolites. However, whether Trichoderma deploys a universal antagonistic program or dynamically adapts its molecular arsenal according to prey identity remains largely unknown. Here, we investigated the phenotypic and transcriptomic responses of four highly antagonistic Trichoderma atroviride strains confronted with damping-off pathogens from three phylogenetically distinct lineages: the ascomycete Alternaria brassicicola, the basidiomycete Rhizoctonia solani, and the oomycete Globisporangium ultimum. In vitro confrontation assays were performed between the four T. atroviride strains and nine pathogen strains (three strains per species). Despite notable intraspecific variation in pathogen sensitivity, all four T. atroviride strains remained highly effective against all tested pathogens. Comparative RNA-seq analyses during direct confrontation with one representative strain per pathogen species revealed strong prey-dependent transcriptional plasticity. Interactions with the two fungal pathogens elicited a robust induction of classical mycoparasitic machinery, including glycoside hydrolases, secreted peptidases, and effector-like proteins, whereas these responses were markedly attenuated in interaction with G. ultimum. Specialized metabolite biosynthetic genes were broadly induced across all interactions, but with largely distinct gene sets depending on the pathogen, with the strongest divergence observed for confrontation with G. ultimum. Beyond this shared response, each fungal pathogen also triggered a distinct host-specific program. In response to A. brassicicola and R. solani, T. atroviride induced distinct sets of genes associated with cell wall organization, while the response to R. solani specifically involved the activation of peroxisomal functions and aromatic amino acid biosynthesis pathways. Receptors potentially involved in pathogen perception also showed pathogen-dependent expression patterns, with distinct GPCRs (G protein-coupled receptors) overexpressed depending on the pathogen encountered. NLRs (NOD-like receptors) displayed a similar pattern, although overexpression was detected only in response to A. brassicicola and G. ultimum. Together, these results demonstrate that T. atroviride does not rely on a fixed mycoparasitic program but instead dynamically remodels its transcriptome according to prey identity, revealing a high degree of transcriptional plasticity underlying broad-spectrum antagonism.

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

Poly(ADP-ribose) polymerases (PARPs) catalyze ADP-ribosylation, a conserved post- translational modification involved in DNA repair, transcriptional regulation, and chromatin remodeling. Although extensively studied in animals, the evolution and diversification of PARPs across the fungal kingdom remain largely unexplored. Here, we present the first kingdom-wide comparative genomic analysis of PARP proteins across 534 fungal species spanning eight phyla. We identified two primary conserved fungal PARP protein types corresponding to human PARP1 and PARP6. Both exhibit highly dynamic evolutionary histories characterized by frequent independent gain and loss events. Ancestral state reconstruction supports the presence of PARP1 in the last common ancestor of fungi, whereas the PARP6-like family has undergone repeated lineage-specific gains and losses. Fungal PARP6-like proteins retain a compact PARP catalytic domain fused to a C-terminal E2 ubiquitin-conjugating domain, whereas the PARP1 family displays extensive structural diversification through domain shuffling and lineage-specific fusions associated with DNA metabolism, chromatin remodeling, and signal transduction. Reconstruction of ancestral catalytic motifs across fungi and other eukaryotes revealed convergent evolution of a non-canonical H-Y-Y catalytic triad, with multiple motif variants co-occurring within individual proteins, suggesting functional diversification. In the Fusarium oxysporum species complex, we identified a lineage-specific expansion of the PARP family, driven exclusively by accessory chromosomes. Genomes with expanded PARP1 repertoires exhibited elevated basal PARylation, increased resistance to DNA-damaging agents that induce single strand breaks, and DNA damage- induced expression of accessory Parp genes. These findings reveal fungal PARPs as evolutionarily dynamic proteins that likely contribute to genome stability, adaptation, and pathogenicity.

Currently, the fungal class Paraglomeromycetes (phylum Glomeromycota) consists of one order, Paraglomerales, with two families: Paraglomeraceae, comprising the genera Paraglomus with ten species and Innospora with one species, and the monogeneric and monospecific Pervetustaceae represented by Pervetustus with Pe. simplex. However, our morphological analyses of these species and an undescribed specimen morphologically resembling other Paraglomeromycetes species, as well as analyses of sequences of these fungi and environmental sequences, indicated that the current taxonomic composition of this class should be updated. We did so here by creating in Paraglomeromycetes Pervetustales ord. nov. with Pervetustus and Opikia catenata gen. nov. and sp. nov., as well as transferring Innospora with In. majewskii to Innosporaceae fam. nov. These novelties were introduced primarily based on strong genetic divergences of the analyzed taxa, as many morphological and histochemical features of their spores and mycorrhizal structures overlap. Only Op. catenata possesses the unique trait of occasionally producing spores in chains. This mode of sporulation has also not been recognized in any other Glomeromycota species. Unlike species of Pervetustales and Innosporaceae, which produce single-walled spores, the subcellular spore structure of Paraglomus species contains one or two walls.

Ada1 (All Development Altered-1) is a conserved but poorly characterised basic leucine zipper (bZIP) transcription factor found throughout filamentous fungi. In the wheat pathogen Parastagonospora nodorum, PnAda1 is required for full virulence and is transcriptionally associated with the virulence regulator PnPf2, but its biological functions remain unclear. Here, we combined comparative RNA sequencing with targeted phenotypic analyses to define the role of PnAda1 during vegetative growth and host infection. Deletion of PnAda1 did not abolish pathogenicity but delayed disease progression, with the PnAda1-deletion mutant transcriptome at 7 days post-inoculation resembling that of the wildtype SN15 at 3 days. This developmental delay was associated with impaired activation of early infection-associated genes, including putative carbohydrate-active enzymes, proteases, transporters and other host-colonisation factors. In contrast, expression of major necrotrophic effector genes was not reduced and instead remained elevated during later stages of infection, indicating that PnAda1 is required for the timely progression of infection-associated transcriptional regulation rather than direct activation of effector genes. Beyond virulence, transcriptomic and phenotypic analyses revealed roles for PnAda1 in nitrogen assimilation, carbon utilisation, abiotic stress responses and fungicide sensitivity. Notably, PnAda1 deletion increased sensitivity to succinate dehydrogenase inhibitor fungicides and reduced expression of succinate dehydrogenase subunit genes. Collectively, our findings identify PnAda1 as a broad regulator of developmental and infection-associated transitions in P. nodorum and expand current understanding of the transcriptional network underlying virulence, metabolism and stress adaptation in an important fungal wheat pathogen.

There is a growing body of research demonstrating the utility of invertebrate models for studies of fungal pathogenesis. In this study we evaluated Tenebrio molitor larvae as a model for studying talaromycosis, the infection caused by Talaromyces marneffei. T. marneffei is a thermally dimorphic opportunistic pathogen of humans, which transitions from hyphae to yeast when exposed to human body temperature (37{degrees}C). Using a combination of virulence assays and histopathology techniques, we have found T. molitor larvae to be useful simple hosts for modelling the yeast-associated disease at 37{degrees}C, as well as for studying the influence of temperature on T. marneffei biology. Infection establishment was found to be temperature-dependent: Larval infections could be established at both 37{degrees}C and 25{degrees}C, however 10-fold higher doses of conidia were required to cause significant disease at 25{degrees}C. Infections were also established more quickly when directly injecting larvae with the yeast cells, indicating that the yeast form has an increased capacity for host damage. T. marneffei in vivo yeast cell development was observed within larval tissues and hemolymph primarily at 37{degrees}C, but also at 25{degrees}C along with filamentous hyphal growth. The larval host environment therefore strongly supports yeast development, even partially in the absence of the 37{degrees}C signal, emphasising the combined importance of temperature and host environment for maintenance of the pathogenic morphology. This work provides a new model to assist future studies of this neglected tropical disease and improves our understanding of the complex relationship between morphology and pathogenicity in T. marneffei.

Fungal diseases cause devastating crop infestations and millions of deaths annually. To improve disease treatment development, we must understand the biological processes these treatments target and the fungal cellular responses they elicit. Current methods to identify genetic responses of fungi to antifungal drugs and fungicides typically involve laborious molecular genetics experiments or transcriptomics. We utilized high-throughput screens in the basidiomycete yeast Rhodotorula (Rhodosporidium) toruloides to rapidly identify genes associated with the fungal response to a copper-zinc fungicide developed by VM Agritech and compared identified genes to those transcriptionally activated in response to the fungicide in R. toruloides and the filamentous ascomycete Neurospora crassa. These 'omics technologies combined with molecular genetics and biochemical analyses showed copper and zinc synergize to inhibit fungal growth due, at least in part, to an increase in cellular zinc concentrations when copper is present. Intriguingly, although we identified genes involved in the copper-zinc fungicide response using both high-throughput screens and transcriptomics, there was little overlap in genes identified through the two methods. Our findings demonstrate the power of combining functional genomics and transcriptomics approaches in identifying a more comprehensive map of the genetic mechanisms necessary for responding to fungicides.

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

Domestication of molds for cheese production has repeatedly shaped Penicillium fungi, most notably giving rise to the emblematic P. camemberti lineage, derived from P. biforme. Here, we identified a new P. biforme lineage, named cheesy, likely selected from P. biforme for food fermentation, including cheese and sausage production. This lineage exhibits evidence of a severe bottleneck, with little nucleotide polymorphism and a single mating type. The cheesy lineage has evolved advantageous traits for cheesemaking: compared to other P. biforme strains and its wild relative P. fuscoglaucum, it displays faster growth on cheese, galactose and lactose media, higher sporulation and germination rates on cheese, elevated lipolytic activity, enhanced inhibition capacities, and produced specific volatile organic compounds. Additionally, P. camemberti and P. biforme cheesy differ in their content of Starship mobile elements, acquired through horizontal transfers. These elements carry cargo genes potentially relevant for adaptation to cheese. Notably, the cheesy lineage has acquired a 20 kb Starship element (Rattus), nested within a much larger 160 kb Starship (Bilge), and carrying cargo genes with predicted functions involved in antagonistic interactions among micro-organisms. SignificanceO_LIWe identified a new Penicillium biforme lineage, named cheesy, which was likely selected from P. biforme for food fermentation (cheese and sausage), genetically and phenotypically different from the emblematic P. camemberti lineage. This lineage suffered from a severe bottleneck, displaying very little nucleotide polymorphism and a single mating type. C_LIO_LIThis newly identified lineage has evolved advantageous traits for cheesemaking; compared to other P. biforme strains and its wild relative P. fuscoglaucum, it displayed faster growth on cheese, galactose and lactose media, higher sporulation and germination rates on cheese, higher lipolytic activity, better inhibition capacities and specific volatile organic compounds. C_LIO_LIPenicillium camemberti and P. biforme cheesy also differed by their content in Starship mobile elements, acquired through horizontal transfers, and carrying cargo genes potentially relevant for adaptation to cheese. C_LIO_LINotably, the cheesy lineage has acquired a specific 20 kb Starship nested within a much larger 160 kb Starship, and likely involved in antagonistic interactions. C_LI

Candida auris (currently Candidozyma auris) is an emerging fungal pathogen responsible for dramatic global increase in invasive candidiasis with high mortality. Most concerning, C. auris has a high propensity to colonize patients and persist and develop multidrug resistance to main classes of antifungals. In this study, we investigated the genetic and phenotypic diversity and resistance mechanisms of C. auris clinical isolates recovered from hospitalized infected patients. A total of 53 isolates from 38 unique patients were recovered from various clinical sources and evaluated for susceptibility to routine antifungal drugs. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis were performed to generate a phylogenetic network to infer population structure and identify mutations associated with drug resistance development. Isolates were also phenotypically evaluated for ability to form biofilms and aggregate, and cell wall adhesins gene expression studies were performed to provide mechanistic insights into C. auris phenotypic plasticity. Except for one clade III isolate, all isolates belonged to clade I and all were resistant to fluconazole with incidence of resistance to amphotericin B, echinocandins or both. Non-synonymous SNPs were found in genes associated with antifungal resistance including ERG11, TAC1B, CDR1 and FKS1. Phenotypically, isolates varied in their ability to form biofilm and aggregate which correlated with expression of the Scf1 and Als4112 cell wall adhesins genes highlighting C. auris phenotypic plasticity in circulating clinical strains. These findings underscore the growing clinical threat posed by C. auris and reinforce the need for optimized surveillance and treatment strategies for controlling its spread.

Recent work has established septation as a critical process for virulence in A. fumigatus, as strains lacking septa are avirulent. While our work has focused on the well-studied Septation Initiation Network (SIN), understanding of septation machinery upstream and downstream of SIN remains limited in filamentous fungi. Proximity labeling techniques are powerful tools to study pathway interactions, especially those that are transient. Recent advances have seen TurboID used to study pathways and processes important for fungal pathogenesis. In this study, we adapt TurboID for use in Aspergillus fumigatus and use overlapping datasets of two SIN components, the terminal NDR kinase SidB and its binding partner MobA, to identify putative septation effectors. Many known septation effectors, including septins and cell wall synthesis proteins, were enriched in our datasets, validating the approach. Phenotypic characterization of hits identified by LC-MS/MS revealed several previously uncharacterized gene products involved in septum formation and cell wall stress tolerance. These included the PP2A regulatory subunit PabA and IQGAP SepG, which have been shown to regulate septation in Aspergillus nidulans. Thus, TurboID is a useful tool to study fungal signaling and physiology in A. fumigatus and may be used to improve understanding of pathogenic processes.

Pseudogymnoascus destructans is a psychrophilic ascomycete fungus that causes White-Nose Syndrome in North American hibernating bats. P. destructans invades the tissue present in bats' noses and wing membranes, causing the characteristic white nose and cupping erosion in the wings. P. destructans exhibits characteristics of a hemibiotrophic fungal pathogen, in which P. destructans can invade the host cells without inducing damage. During this stage, P. destructans hyphae assume a morphology similar to fungal plant pathogens during the biotrophic stage of infection. However, the mechanism by which P. destructans regulates its morphology and virulence is not fully understood. The phosphatase calcineurin is a key regulator of fungal stress response and virulence and could play a role in P. destructans virulence. Calcineurin can be inhibited using FK506 or Cyclosporin A (CSA). FK506 interacts with FKBP12, while CSA interacts with Cyclosporin A, and the protein-drug complex then inhibits calcineurin activity. Here, we show that P. destructans is intrinsically resistant to one calcineurin inhibitor (FK506), possibly because P. destructans FKBP12 has a lysine instead of the conserved arginine at position 89, which has been shown to reduce the affinity of the FKBP12-FK506 complex for calcineurin. Using cyclosporin A, we found that calcineurin is important for fungal growth. Additionally, calcineurin inhibition increases voriconazole inhibition. In contrast, calcineurin inhibition was antagonistic to the anti-cell wall drug micafungin. Taken together, calcineurin's role in fungal growth and response to voriconazole is conserved in P. destructans but may play a unique role in P. destructans response to micafungin.

Cutaneotrichosporon dermatis (formerly Trichosporon dermatis) is a basidiomycetous yeast-like fungus known to cause summer-type hypersensitivity pneumonitis, although its virulence in humans remains poorly understood. We performed morphological and molecular identification of an isolate from the sputum and blood cultures of an immunocompromised patient, together with pathogenicity assessment using a Galleria mellonella model, biofilm formation/eradication assays, antifungal susceptibility testing, drug combination effects, and the post-antifungal effect (PAFE), compared with Trichosporon asahii. The isolate was identified as C. dermatis by ITS/IGS1 sequencing, supported by phylogenetic analysis. Growth of C. dermatis increased more at 37 than at 25. In the Galleria mellonella assay, C. dermatis, T. asahii, and Candida albicans each showed dose-dependent pathogenicity at sufficiently high inocula, although Rhizopus oryzae was the most potent pathogen on a per-CFU basis. C. dermatis formed biofilms that were more completely inhibited by terbinafine (TRB) and amphotericin B (AmB) than azole agents, which showed only partial inhibitory activity even at high concentrations. Susceptibility testing showed relatively strong susceptibility to AmB and azole agents. In the TRB and azole combination assay, the fractional inhibitory concentration index (FICI) was below 0.5, indicating synergy. Isavuconazole (ISC) showed a markedly stronger PAFE than the other azole agents tested. These findings indicate that although azoles show only partial efficacy against its biofilm, C. dermatis can still cause invasive infection, and that azole monotherapy or TRB and azole combination therapy, aided by the potent PAFE of ISC, may represent effective treatment options.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

PurposeElucidate the influence of glucose metabolic pathways on A. fumigatus lung and corneal infection. MethodsThe A. fumigatus acuF and creA genes were deleted in an mcherry-expressing strain. The mutants were tested for alterations in radial growth, cell wall composition by fluorescence staining assays, and antifungal sensitivity through broth microdilution assays. Hyphal penetration of the strains through explanted porcine corneas was tracked by confocal microscopy using the mCherry signal. Virulence was evaluated in established models of invasive pulmonary aspergillosis (IPA) and fungal keratitis (FK) using C57BL/6J mice. ResultsDeletion of the A. fumigatus phosphoenolpyruvate carboxykinase (acuF) resulted in a dependency on exogenous glucose to support growth in vitro, but did not impact virulence in either the IPA or FK models. Loss of the carbon catabolite repressor CreA resulted in a broad dysregulation of carbon metabolic pathways and altered cell wall homeostasis. Surprisingly, whereas the{Delta} creA remained fully virulent in the lung, the mutant was unable to establish infection in the FK model. This in vivo phenotype corresponded to an inability of{Delta} creA to physically invade porcine corneal explants, which we attributed to a marked reduction in cell wall chitin content. ConclusionsGluconeogenesis is dispensable for A. fumigatus lung and corneal infection, suggesting tissue-derived glucose supports fungal growth in both environments. Loss of CreA disrupts glucose assimilation, its synthesis into chitin and, consequently, cell rigidity and hyphal invasion into the dense corneal stroma. Thus, CreA and other cell wall regulatory proteins may serve as targets for novel FK antifungals.

Invasive fungal infections are on the rise due to climate change, antifungal resistance, and increased usage of immunomodulating therapies. The antifungal arsenal is limited in number and in efficacy necessitating novel therapeutics. Deletion of the gene encoding the fungal yeast casein kinase, YCK2, has pleiotropic effects, impacting morphology, drug resistance, metabolism, cell wall, and virulence. Yck2 has recently been shown to be druggable, however the mechanisms by which Yck2 elicits its impacts are unknown. We show that C. neoformans Yck2 is a regulator of cell wall masking, thermotolerance, general stress, and drug resistance. Proximity labeling of Yck2-interacting proteins revealed that Yck2 interacts with proteins that function in the mitochondria as well as the essential plasma membrane H+ ATPase, Pma1. A role for Yck2 in mitochondrial regulation was supported by sensitivity to mitochondrial inhibitors and increased mitochondrial ROS production in the absence of Yck2, and presence of Yck2 in mitochondrial fractions. Using pHluorin-expressing cells we also found that intracellular pH is elevated in cells lacking Yck2 supporting a role in regulating Pma1 activity. Both mitochondria and cellular pH may contribute to cellular signaling and may explain the multitude of phenotypes associated with the yck2{Delta} mutant. Our results begin to identify the mechanisms by which Yck2 contributes to fungal cellular homeostasis which may support efforts to optimize Yck2 inhibitors.

Budding yeast cells can transition from vegetative to filamentous growth under specific environmental stimuli. Saccharomyces cerevisiae has served as a model to understand this process, which is linked to the virulence of many fungal pathogens. One manifestation of filamentation in S. cerevisiae is the formation of pseudohyphal cells, which are elongated and divide in a polarized manner. Here, we established a live-cell imaging assay to monitor this morphogenetic transition and developed a set of fluorescent expression reporters to probe the activity of various signaling pathways during the first 10 hours of this transition. This approach allowed us to identify the stimulus that activates the filamentous growth Mitogen-Activated Protein Kinase (fgMAPK) pathway, which has long evaded identification. Our results indicate that the fgMAPK cascade senses mechanical cues and becomes activated when cells grow on a solid surface. In addition, we have observed that the stimulation of the transcription factor Gln3 regulated by the TOR pathway in low ammonium condition correlates with the elongation of the cells.

Candida auris is an emerging multidrug-resistant fungal pathogen. However, little is known about the role of long noncoding RNAs (lncRNAs) in the human immune response to this fungus. We reanalyzed RNA-sequencing data from peripheral blood mononuclear cells obtained from three healthy donors. The cells were exposed to live C. auris, purified mannan, purified {beta} glucan, or RPMI control for 4 h and 24 h. We identified 607 putative novel polyadenylated lncRNA loci that were absent from GENCODE Release 48. Of the 449 novel loci tested for differential expression, 102 responded to at least one stimulus. Novel-lncRNA responses were limited at 4 h but increased markedly at 24 h. At 24 h, 78 novel lncRNAs responded to live C. auris, 34 to mannan, and 11 to {beta}-glucan. Many of these responses were specific to a particular stimulus. Among the 102 responsive lncRNAs, 94 were significantly co-expressed with protein coding genes. The coding-gene partners of the most highly connected lncRNAs were associated with innate immunity, defense responses, lysosomal functions, and signaling pathways. Temporal analysis identified 19 novel lncRNAs whose responses changed between 4 and 24 h. Fourteen were also differentially expressed and were selected for further analysis. Combining temporal response, co-expression, and genomic proximity identified 4 candidate cis-associated pairs: MSTRG.32011-TLR2, MSTRG.5763-DUSP5, MSTRG.25627-HCK, and MSTRG.18159 ITGB3.These findings reveal a predominantly late and stimulus-specific novel-lncRNA response to C. auris and provide prioritized candidates for experimental validation.