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Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

Pathogens must efficiently acquire nutrients from host tissue to proliferate, and strategies to block pathogen access therefore hold promise for disease control. In this study, we investigated whether heme biosynthesis is an effective target for ablating the virulence of the phytopathogenic fungus Ustilago maydis on maize plants. We first constructed conditional heme auxotrophs of the fungus by placing the heme biosynthesis gene hem12 encoding uroporphyrinogen decarboxylase (Urod) under the control of nitrogen or carbon source-regulated promoters. These strains were heme auxotrophs under non-permissive conditions and unable to cause disease in maize seedlings, thus demonstrating the inability of the fungus to acquire sufficient heme from host tissue to support proliferation. Subsequent experiments characterized the role of endocytosis in heme uptake, the susceptibility of the fungus to heme toxicity as well as the transcriptional response to exogenous heme. The latter RNA-seq experiments identified a candidate ABC transporter with a role in the response to heme and xenobiotics. Given the importance of heme biosynthesis for U. maydis pathogenesis, we tested the ability of the well-characterized herbicide BroadStar to influence disease. This herbicide contains the active ingredient flumioxazin, an inhibitor of Hem14 in the heme biosynthesis pathway, and we found that it was an effective antifungal agent for blocking disease in maize. Thus, repurposing herbicides for which resistant plants are available may be an effective strategy to control pathogens and achieve crop protection., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Fungi employ diverse mechanisms for iron uptake to ensure proliferation and survival in iron-limited environments. Siderophores are secondary metabolite small molecules with a high affinity specifically for ferric iron; these molecules play an essential role in iron acquisition in fungi and significantly influence fungal physiology and virulence. Fungal siderophores, which are primarily hydroxamate types, are synthesized via non-ribosomal peptide synthetases (NRPS) or NRPS-independent pathways. Following synthesis, siderophores are excreted, chelate iron, and are transported into the cell by specific cell membrane transporters. In several human pathogenic fungi, siderophores are pivotal for virulence, as inhibition of their synthesis or transport significantly reduces disease in murine models of infection. This review briefly highlights siderophore biosynthesis and transport mechanisms in fungal pathogens as well the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe . Understanding siderophore biosynthesis and transport in pathogenic fungi provides valuable insights into fungal biology and illuminates potential therapeutic targets for combating fungal infections.

There is an urgent need for new antifungal drugs to treat invasive fungal diseases. Unfortunately, the echinocandin drugs that are fungicidal against other important fungal pathogens are ineffective against Cryptococcus neoformans , the causative agent of life-threatening meningoencephalitis in immunocompromised people. Contributing mechanisms for echinocandin tolerance are emerging with connections to calcineurin signaling, the cell wall, and membrane composition. In this context, we discovered that a defect in phosphate uptake impairs the tolerance of C. neoformans to the echinocandin caspofungin. Our previous analysis of mutants lacking three high affinity phosphate transporters revealed reduced elaboration of the polysaccharide capsule and attenuated virulence in mice. We investigated the underlying mechanisms and found that loss of the transporters and altered phosphate availability influences the cell wall and membrane composition. These changes contribute to the shedding of capsule polysaccharide thus explaining the reduced size of capsules on mutants lacking the phosphate transporters. We also found an influence of the calcineurin pathway including calcium sensitivity and an involvement of the endoplasmic reticulum in the response to phosphate limitation. Furthermore, we identified membrane and lipid composition changes consistent with the role of phosphate in phospholipid biosynthesis and with previous studies implicating membrane integrity in caspofungin tolerance. Finally, we discovered a contribution of phosphate to titan cell formation, a cell type that displays modified cell wall and capsule composition. Overall, our analysis reinforces the importance of phosphate as a regulator of cell wall and membrane composition with implications for capsule attachment and antifungal drug susceptibility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Qu, Bhalla, Horianopoulos, Hu, Alcázar Magaña, Foster, Roque da Silva, Kretschmer and Kronstad.)

The basidiomycete fungus Cryptococcus neoformans is a useful model for investigating mechanisms of fungal pathogenesis in mammalian hosts. This pathogen is the causative agent of cryptococcal meningitis in immunocompromised patients and is in the critical priority group of the World Health Organization fungal priority pathogens list. In this study, we employed a mutant lacking the OPI3 gene encoding a methylene-fatty-acyl-phospholipid synthase to characterize the role of phosphatidylcholine (PC) and lipid homeostasis in the virulence of C. neoformans . We first confirmed that OPI3 was required for growth in nutrient limiting conditions, a phenotype that could be rescued with exogenous choline and PC. Additionally, we established that loss of Opi3 and the lack of PC lead to an accumulation of neutral lipids in lipid droplets and alterations in major lipid classes. The growth defect of the opi3Δ mutant was also rescued by sorbitol and polyethylene glycol (PEG), a result consistent with protection of ER function from the stress caused by lipid imbalance. We then examined the impact of Opi3 on virulence and found that the dependence of PC synthesis on Opi3 caused reduced capsule size and this was accompanied by an increase in shed capsule polysaccharide and changes in cell wall composition. Further tests of virulence demonstrated that survival in alveolar macrophages and the ability to cause disease in mice were not impacted by loss of Opi3 despite the choline auxotrophy of the mutant in vitro . Overall, this work establishes the contribution of lipid balance to virulence factor elaboration by C. neoformans and suggests that host choline is sufficient to support proliferation during disease., Competing Interests: FM is an employee of Springer Nature. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lee, Brisland, Qu, Horianopoulos, Hu, Mayer and Kronstad.)

The fungal pathogen Cryptococcus neoformans is well adapted to its host environment. It has several defence mechanisms to evade oxidative and nitrosative agents released by phagocytic host cells during infection. Among them, melanin production is linked to both fungal virulence and defence against harmful free radicals that facilitate host innate immunity. How C. neoformans manipulates its redox environment to facilitate melanin formation and virulence is unclear. Here we show that the antioxidant glutathione is inextricably linked to redox-active processes that facilitate melanin and titan cell production, as well as survival in macrophages and virulence in a murine model of cryptococcosis. Comparative metabolomics revealed that disruption of glutathione biosynthesis leads to accumulation of reducing and acidic compounds in the extracellular environment of mutant cells. Overall, these findings highlight the importance of redox homeostasis and metabolic compensation in pathogen adaptation to the host environment and suggest new avenues for antifungal drug development., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Small GTPases of the Rab family coordinate multiple membrane fusion and trafficking events in eukaryotes. In fungi, the Rab GTPase, Ypt7, plays a critical role in late endosomal trafficking, and is required for homotypic fusion events in vacuole biogenesis and inheritance. In this study, we identified a putative YPT7 homologue in Cryptococcus neoformans , a fungal pathogen causing life threatening meningoencephalitis in immunocompromised individuals. As part of an ongoing effort to understand mechanisms of iron acquisition in C. neoformans , we established a role for Ypt7 in growth on heme as the sole iron source. Deletion of YPT7 also caused abnormal vacuolar morphology, defective endocytic trafficking and autophagy, and mislocalization of Aph1, a secreted vacuolar acid phosphatase. Ypt7 localized to the vacuolar membrane and membrane contact sites between the vacuole and mitochondria (vCLAMPs), and loss of the protein impaired growth on inhibitors of the electron transport chain. Additionally, Ypt7 was required for robust growth at 39°C, a phenotype likely involving the calcineurin signaling pathway because ypt7 mutants displayed increased susceptibility to the calcineurin-specific inhibitors, FK506 and cyclosporin A; the mutants also had impaired growth in either limiting or high levels of calcium. Finally, Ypt7 was required for survival during interactions with macrophages, and ypt7 mutants were attenuated for virulence in a mouse inhalation model thus demonstrating the importance of membrane trafficking functions in cryptococcosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hu, Qu, Bhalla, Xue, Bakkeren, Lee and Kronstad.)

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans . BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae , BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans . BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis., Importance: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii . There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans . BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans , acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans . We propose BRI as a new antifungal agent against cryptococcosis., Competing Interests: Gustavo H. Goldman is a member of the scientific advisory board of Innovation Pharmaceuticals, a company that is conducting clinical trials on brilacidin. Other authors have no conflict of interest.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans .IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans . This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi., Competing Interests: The authors declare no conflict of interest.

The corn smut fungus, Ustilago maydis , is an excellent model for studying biotrophic plant-pathogen interactions, including nutritional adaptation to the host environment. Iron acquisition during host colonization is a key aspect of microbial pathogenesis yet less is known about this process for fungal pathogens of plants. Monothiol glutaredoxins are central regulators of key cellular functions in fungi, including iron homeostasis, cell wall integrity, and redox status via interactions with transcription factors, iron-sulfur clusters, and glutathione. In this study, the roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis were investigated by constructing strains expressing a conditional allele of grx4 under the control of the arabinose-inducible, glucose-repressible promoter P crg 1 . The use of conditional expression was necessary because Grx4 appeared to be essential for U. maydis. Transcriptome and genetic analyses with strains depleted in Grx4 revealed that the protein participates in the regulation of iron acquisition functions and is necessary for the ability of U. maydis to cause disease on maize seedlings. Taken together, this study supports the growing appreciation of monothiol glutaredoxins as key regulators of virulence-related phenotypes in pathogenic fungi.

The current therapeutic challenges for treating fungal diseases demand new approaches and new drugs. A promising strategy involves combination therapy with agents of distinct mechanisms of action to increase fungicidal activity and limit the impact of mutations leading to resistance. In this study, we evaluated the antifungal potential of bortezomib by examining the inhibition of proteasome activity, cell proliferation, and capsule production by Cryptococcus neoformans , the causative agent of fungal meningoencephalitis. Chemical genetic screens with collections of deletion mutants identified potential druggable targets for combination therapy with bortezomib. In vitro assays of combinations of bortezomib with flucytosine, chlorpromazine, bafilomycin A1, copper sulfate, or hydroxyurea revealed antifungal effects against C. neoformans . Furthermore, combination treatment with bortezomib and flucytosine in a murine inhalation model of cryptococcosis resulted in the improvement of neurological functions and reduced fungal replication and dissemination, leading to a delay in disease progression. This study therefore highlights the utility of chemical genetic screens to identify new therapeutic approaches as well as the antifungal potential of proteasome inhibition. IMPORTANCE Fungal diseases of humans are difficult to treat, and there is a clear need for additional antifungal drugs, better diagnostics, effective vaccines, and new approaches to deal with emerging drug resistance. Fungi are challenging to control because they share many common biochemical functions with their mammalian hosts and it is therefore difficult to identify fungal-specific targets for drug development. One approach is to employ existing antifungal drugs in combination with agents that target common cellular processes at levels that are (ideally) not toxic for the host. We pursued this approach in this study by examining the potential of the clinically approved proteasome inhibitor bortezomib to influence the proliferation and virulence of Cryptococcus neoformans . We found that the combination of bortezomib with the anti-cryptococcal drug flucytosine improved the survival of infected mice, thus demonstrating the potential of this strategy for antifungal therapy.

Recent studies in pathogenic yeasts reinforce our appreciation of the influence of metal homeostasis on the fungal cell surface. To illustrate this influence, we focus on recent studies on Cryptococcus neoformans, a fungal pathogen with a complex surface of a cell wall with embedded melanin and an attached polysaccharide capsule. Copper and iron are essential yet toxic metals, and current efforts demonstrate the importance of these metals for modulating the surface structure of C. neoformans cells in ways that contribute to fungal-host interactions during disease in vertebrate hosts. In this review, we briefly summarize mechanisms of acquisition and regulation for copper and iron, and then discuss recent insights into the connections between the metals and the cell surface., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cryptococcus gattii and Cryptococcus neoformans are the main etiological agents of cryptococcosis, an invasive mycosis treated with amphotericin B, 5-fluorocytosine, and fluconazole. This limited arsenal is toxic and is associated with antifungal resistance. Cryptococcosis and malaria pathogens are eukaryotic organisms that have a high incidence in Sub-Saharan Africa. The antimalarials (ATMs) halofantrine (HAL) and amodiaquine (AQ) block Plasmodium heme polymerase, and artesunate (ART) induces oxidative stress. Considering that Cryptococcus spp. is susceptible to reactive oxygen species and that iron is essential for metabolism, the repurposing of ATMs for treating cryptococcosis was tested. ATMs reduced fungal growth, induced oxidative and nitrosative stresses, and altered ergosterol content, melanin production, and polysaccharide capsule size in C. neoformans and C. gattii, revealing a dynamic effect on fungal physiology. A comprehensive chemical-genetic analysis using two mutant libraries demonstrated that the deletion of genes involved in synthesizing components of the plasma membrane and cell wall, and oxidative stress responses are essential for fungal susceptibility to ATMs. Interestingly, the amphotericin B (AMB) fungicidal concentrations were ∼10 times lower when combined with ATMs, demonstrating a synergistic interaction. Further, the combinations showed reduced toxicity to murine macrophages. Finally, HAL+AMB and AQ+AMB efficiently reduced lethality and fungal burden in the lungs and brain in murine cryptococcosis. These findings provide perspectives for further studies with ATMs against cryptococcosis and other fungal infections., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Fungal pathogens cause life-threatening diseases in humans, and the increasing prevalence of these diseases emphasizes the need for new targets for therapeutic intervention. Nutrient acquisition during infection is a promising target, and recent studies highlight the contributions of endomembrane trafficking, mitochondria, and vacuoles in the sensing and acquisition of heme by fungi. These studies have been facilitated by genetically encoded biosensors and other tools to quantitate heme in subcellular compartments and to investigate the dynamics of trafficking in living cells. In particular, the applications of biosensors in fungi have been extended beyond the detection of metabolites, cofactors, pH, and redox status to include the detection of heme. Here, we focus on studies that make use of biosensors to examine mechanisms of heme uptake and degradation, with guidance from the model fungus Saccharomyces cerevisiae and an emphasis on the pathogenic fungi Candida albicans and Cryptococcus neoformans that threaten human health. These studies emphasize a role for endocytosis in heme uptake, and highlight membrane contact sites involving mitochondria, the endoplasmic reticulum and vacuoles as mediators of intracellular iron and heme trafficking., Competing Interests: Declaration of competing interest The authors declare that no competing interests exist.

Verticillium transcription activator of adhesion 3 (Vta3) is required for plant root colonization and pathogenicity of the soil-borne vascular fungus Verticillium dahliae. RNA sequencing identified Vta3-dependent genetic networks required for growth in tomato xylem sap. Vta3 affects the expression of more than 1,000 transcripts, including candidates with predicted functions in virulence and morphogenesis such as Egh16-like virulence factor 1 (Elv1) and Master transcription factor 1 (Mtf1). The genes encoding Elv1 and Mtf1 were deleted and their functions in V. dahliae growth and virulence on tomato (Solanum lycopersicum) plants were investigated using genetics, plant infection experiments, gene expression studies and phytohormone analyses. Vta3 contributes to virulence by promoting ELV1 expression, which is dispensable for vegetative growth and conidiation. Vta3 decreases disease symptoms mediated by Mtf1 in advanced stages of tomato plant colonization, while Mtf1 induces the expression of fungal effector genes and tomato pathogenesis-related protein genes. The levels of pipecolic and salicylic acids functioning in tomato defense signaling against (hemi-) biotrophic pathogens depend on the presence of MTF1, which promotes the formation of resting structures at the end of the infection cycle. In summary, the presence of VTA3 alters gene expression of virulence factors and tames the Mtf1 genetic subnetwork for late stages of plant disease progression and subsequent survival of the fungus in the soil., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Maurus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The phenotypic plasticity of Cryptococcus neoformans is widely studied and demonstrated in vitro, but its influence on pathogenicity remains unclear. In this study, we investigated the dynamics of cryptococcal cell and transcriptional remodeling during pulmonary infection in a murine model. We showed that in Cryptococcus neoformans, cell size reduction (cell body ≤ 3 µm) is important for initial adaptation during infection. This change was associated with reproductive fitness and tissue invasion. Subsequently, the fungus develops mechanisms aimed at resistance to the host’s immune response, which is determinant for virulence. We investigated the transcriptional changes involved in this cellular remodeling and found an upregulation of transcripts related to ribosome biogenesis at the beginning (6 h) of infection and a later (10 days) upregulation of transcripts involved in the inositol pathway, energy production, and the proteasome. Consistent with a role for the proteasome, we found that its inhibition delayed cell remodeling during infection with the H99 strain. Altogether, these results further our understanding of the infection biology of C. neoformans and provide perspectives to support therapeutic and diagnostic targets for cryptococcosis.

Verticillium dahliae causes economic losses to a wide range of crops as a vascular fungal pathogen. This filamentous ascomycete spends long periods of its life cycle in the plant xylem, a unique environment that requires adaptive processes. Specifically, fungal proteins produced in the xylem sap of the plant host may play important roles in colonizing the plant vasculature and in inducing disease symptoms. RNA sequencing revealed over 1500 fungal transcripts that are significantly more abundant in cells grown in tomato xylem sap compared with pectin-rich medium. Of the 85 genes that are strongly induced in the xylem sap, four genes encode the hydrophobins Vdh1, Vdh2, Vdh4 and Vdh5. Vdh4 and Vhd5 are structurally distinct from each other and from the three other hydrophobins (Vdh1-3) annotated in V. dahliae JR2. Their functions in the life cycle and virulence of V. dahliae were explored using genetics, cell biology and plant infection experiments. Our data revealed that Vdh4 and Vdh5 are dispensable for V. dahliae development and stress response, while both contribute to full disease development in tomato plants by acting at later colonization stages. We conclude that Vdh4 and Vdh5 are functionally specialized fungal hydrophobins that support pathogenicity against plants.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Ferritin, a major iron storage protein in vertebrates, supplies iron upon iron deficiency. Ferritin is also found extracellularly, and acts as an iron carrier and a contributor to the immune response to invading microbes. Some microbial pathogens take advantage of ferritin as an iron source upon infection. However, no information is currently available on whether the human fungal pathogen Cryptococcus neoformans can acquire iron from ferritin. Here, we found that C. neoformans grew well in the presence of ferritin as a sole iron source. We showed that the binding of ferritin to the surface of C. neoformans is necessary and that acidification may contribute to ferritin-iron utilization by the fungus. Our data also revealed that the high-affinity reductive iron uptake system in C. neoformans is required for ferritin-iron acquisition. Furthermore, phagocytosis of C. neoformans by macrophages led to increased intracellular ferritin levels, suggesting that iron is sequestered by ferritin in infected macrophages. The increase in intracellular ferritin levels was reversed upon infection with a C. neoformans mutant deficient in the high-affinity reductive iron uptake system, indicating that this system plays a major role in iron acquisition in the phagocytosed C. neoformans in macrophages., Lay Summary: Cryptococcus neoformans is an opportunistic fungal pathogen causing life-threatening pulmonary disease and cryptococcal meningitis, mainly in immunocompromised patients. In this study, we found that C. neoformans can use ferritin, a major iron storage protein in vertebrates, as a sole iron source., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Phosphate is an essential macronutrient for fungal proliferation as well as a key mediator of antagonistic, beneficial, and pathogenic interactions between fungi and other organisms. In this review, we summarize recent insights into the integration of phosphate metabolism with mechanisms of fungal adaptation that support growth and survival. In particular, we highlight aspects of phosphate sensing important for responses to stress and regulation of cell-surface changes with an impact on fungal pathogenesis, host immune responses, and disease outcomes. Additionally, new studies provide insights into the influence of phosphate availability on cooperative or antagonistic interactions between fungi and other microbes, the associations of mycorrhizal and endophytic fungi with plants, and connections with plant immunity. Overall, phosphate homeostasis is emerging as an integral part of fungal metabolism and communication to support diverse lifestyles., Competing Interests: Declaration of interests There are no interests to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Histone chaperoning ensures genomic integrity during routine processes such as DNA replication and transcription as well as DNA repair upon damage. Here, we identify a nuclear J domain protein, Dnj4, in the fungal pathogen Cryptococcus neoformans and demonstrate that it interacts with histones 3 and 4, suggesting a role as a histone chaperone. In support of this idea, a dnj4Δ deletion mutant had elevated levels of DNA damage and was hypersensitive to DNA-damaging agents. The transcriptional response to DNA damage was also impaired in the dnj4Δ mutant. Genes related to DNA damage and iron homeostasis were upregulated in the wild-type strain in response to hydroxyurea treatment; however, their upregulation was either absent from or reduced in the dnj4Δ mutant. Accordingly, excess iron rescued the mutant's growth in response to DNA-damaging agents. Iron homeostasis is crucial for virulence in C. neoformans; however, Dnj4 was found to be dispensable for disease in a mouse model of cryptococcosis. Finally, we confirmed a conserved role for Dnj4 as a histone chaperone by expressing it in Saccharomyces cerevisiae and showing that it disrupted endogenous histone chaperoning. Altogether, this study highlights the importance of a JDP cochaperone in maintaining genome integrity in C. neoformans. IMPORTANCE DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones. In this study, we show that a histone chaperone, Dnj4, is required for genome integrity and for the response to DNA damage. The gene encoding this protein in Cryptococcus neoformans lacks an ortholog in Saccharomyces cerevisiae; however, it is conserved in humans in which its ortholog is essential. Since it is not essential in C. neoformans, we were able to generate deletion mutants to characterize the roles of Dnj4. We also expressed Dnj4 in S. cerevisiae, in which it was able to bind S. cerevisiae histones and interfere with existing histone chaperoning machinery. Therefore, we show a conserved role for Dnj4 in histone chaperoning that suggests that C. neoformans is useful to better understand aspects of this important biological process.

The pathogenic fungus Cryptococcus neoformans must overcome iron limitation to cause disease in mammalian hosts. Previously, we reported a screen for insertion mutants with poor growth on haem as the sole iron source. In this study, we characterised one such mutant and found that the defective gene encoded a Vam6/Vps39/TRAP1 domain-containing protein required for robust growth on haem, an important iron source in host tissue. We designated this protein Vps3 based on reciprocal best matches with the corresponding protein in Saccharomyces cerevisiae. C. neoformans encodes a second Vam6/Vps39/TRAP1 domain-containing protein designated Vam6/Vlp1, and we found that this protein is also required for robust growth on haem as well as on inorganic iron sources. This protein is predicted to be a component of the homotypic fusion and vacuole protein sorting complex involved in endocytosis. Further characterisation of the vam6Δ and vps3Δ mutants revealed perturbed trafficking of iron acquisition functions (e.g., the high affinity iron permease Cft1) and impaired processing of the transcription factor Rim101, a regulator of haem and iron acquisition. The vps3Δ and vam6Δ mutants also had pleiotropic phenotypes including loss of virulence in a mouse model of cryptococcosis, reduced virulence factor elaboration and increased susceptibility to stress, indicating pleiotropic roles for Vps3 and Vam6 beyond haem use in C. neoformans. TAKE AWAYS: Two Vam6/Vps39/TRAP1-domain proteins, Vps3 and Vam6, support the growth of Cryptococcus neoformans on haem. Loss of Vps3 and Vam6 influences the trafficking and expression of iron uptake proteins. Loss of Vps3 or Vam6 eliminates the ability of C. neoformans to cause disease in a mouse model of cryptococcosis., (© 2021 John Wiley & Sons Ltd.)

Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here, we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37°C and 39°C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white (CFW), and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the CWI pathway in cells grown at elevated temperature or upon treatment with CFW, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ, and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall, and other stresses. In summary, we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The capacity of opportunistic fungal pathogens such as Cryptococcus neoformans to cause disease is dependent on their ability to overcome an onslaught of stresses including elevated temperature under mammalian host conditions. Protein chaperones and co-chaperones play key roles in thermotolerance. In this study, we characterized the role of the endoplasmic reticulum (ER) J-domain containing co-chaperone, Dnj1, in the virulence of C. neoformans . A strain expressing a Dnj1-GFP fusion protein was used to confirm localization to the ER, and a dnj1∆ deletion mutant was shown to be hypersensitive to the ER stress caused by tunicamycin (TM) or 4μ8C. Dnj1 and another ER chaperone, calnexin were found to coordinately maintain ER homeostasis and contribute to maintenance of cell wall architecture. Dnj1 also contributed to thermotolerance and increased in abundance at elevated temperatures representative of febrile patients (e.g., 39°C) thus highlighting its role as a temperature-responsive J domain protein. The elaboration of virulence factors such as the polysaccharide capsule and extracellular urease activity were also markedly impaired in the dnj1∆ mutant when induced at human body temperature (i.e., 37°C). These virulence factors are immunomodulatory and, indeed, infection with the dnj1∆ mutant revealed impaired induction of the cytokines IL-6, IL-10, and MCP-1 in the lungs of mice compared to infection with wild type or complemented strains. The dnj1∆ mutant also had attenuated virulence in an intranasal murine model of cryptococcosis. Altogether, our data indicate that Dnj1 is crucial for survival and virulence factor production at elevated temperatures. The characterization of this co-chaperone also highlights the importance of maintaining homeostasis in the ER for the pathogenesis of C. neoformans ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Horianopoulos, Lee, Hu, Caza and Kronstad.)

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Iron acquisition is critical for pathogenic fungi to adapt to and survive within the host environment. However, to same extent, the fungi must also avoid the detrimental effects caused by excess iron. The importance of iron has been demonstrated for the physiology and virulence of major fungal pathogens of humans including Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. In particular, numerous studies have revealed that aspects of iron acquisition, metabolism, and homeostasis in the fungal pathogens are tightly controlled by conserved transcriptional regulators including a GATA-type iron transcription factor and the CCAAT-binding complex (CBC)/HapX orthologous protein complex. However, the specific downstream regulatory networks are slightly different in each fungus. In addition, roles have been proposed or demonstrated for other factors including monothiol glutaredoxins, BolA-like proteins, and Fe-S cluster incorporation on the GATA-type iron transcription factor and the CBC/HapX orthologous protein complex, although limited information is available. Here we focus on recent work on C. neoformans in the context of an emerging framework for fungal regulation of iron acquisition, metabolism, and homeostasis. Our specific goal is to summarize recent findings on transcriptional networks governed by the iron regulators Cir1 and HapX in C. neoformans.

Competing Interests: "The authors have declared that no competing interests exist."

Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2 , which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug fluconazole caused increased vacuolar fragmentation. These conditions also commonly cause an increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly due to copper or iron limitation.

Differentiation, growth, and virulence of the vascular plant pathogen Verticillium dahliae depend on a network of interconnected cellular signaling cascades. The transcription factor Hac1 of the endoplasmic reticulum-associated unfolded protein response (UPR) is required for initial root colonization, fungal growth, and vascular propagation by conidiation. Hac1 is essential for the formation of microsclerotia as long-time survival resting structures in the field. Single endoplasmic reticulum-associated enzymes for linoleic acid production as precursors for oxylipin signal molecules support fungal growth but not pathogenicity. Microsclerotia development, growth, and virulence further require the pheromone response mitogen-activated protein kinase (MAPK) pathway, but without the Ham5 scaffold function. The MAPK phosphatase Rok1 limits resting structure development of V. dahliae , but promotes growth, conidiation, and virulence. The interplay between UPR and MAPK signaling cascades includes several potential targets for fungal growth control for supporting disease management of the vascular pathogen V. dahliae .

The heat shock proteins (HSPs) function as chaperones to facilitate proper folding and modification of proteins and are of particular importance when organisms are subjected to unfavourable conditions. The human fungal pathogens are subjected to such conditions within the context of infection as they are exposed to human body temperature as well as the host immune response. Herein, the roles of the major classes of HSPs are briefly reviewed and their known contributions in human fungal pathogens are described with a focus on Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus . The Hsp90s and Hsp70s in human fungal pathogens broadly contribute to thermotolerance, morphological changes required for virulence, and tolerance to antifungal drugs. There are also examples of J domain co-chaperones and small HSPs influencing the elaboration of virulence factors in human fungal pathogens. However, there are diverse members in these groups of chaperones and there is still much to be uncovered about their contributions to pathogenesis. These HSPs do not act in isolation, but rather they form a network with one another. Interactions between chaperones define their specific roles and enhance their protein folding capabilities. Recent efforts to characterize these HSP networks in human fungal pathogens have revealed that there are unique interactions relevant to these pathogens, particularly under stress conditions. The chaperone networks in the fungal pathogens are also emerging as key coordinators of pathogenesis and antifungal drug tolerance, suggesting that their disruption is a promising strategy for the development of antifungal therapy.

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans , a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii . A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans . Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans .

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae . Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1 , NLP2 , and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization., (Copyright © 2020 Leonard, Kühn, Harting, Maurus, Nagel, Starke, Kusch, Valerius, Feussner, Feussner, Kaever, Landesfeind, Morgenstern, Becher, Hecker, Braus-Stromeyer, Kronstad and Braus.)

Iron is essential for the growth of the human fungal pathogen Cryptococcus neoformans within the vertebrate host, and iron sensing contributes to the elaboration of key virulence factors, including the formation of the polysaccharide capsule. C. neoformans employs sophisticated iron acquisition and utilization systems governed by the transcription factors Cir1 and HapX. However, the details of the transcriptional regulatory networks that are governed by these transcription factors and connections to virulence remain to be defined. Here, we used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome analysis (RNA-seq) to identify genes directly regulated by Cir1 and/or HapX in response to iron availability. Overall, 40 and 100 genes were directly regulated by Cir1, and 171 and 12 genes were directly regulated by HapX, under iron-limited and replete conditions, respectively. More specifically, we found that Cir1 directly controls the expression of genes required for iron acquisition and metabolism, and indirectly governs capsule formation by regulating specific protein kinases, a regulatory connection not previously revealed. HapX regulates the genes responsible for iron-dependent pathways, particularly under iron-depleted conditions. By analyzing target genes directly bound by Cir1 and HapX, we predicted the binding motifs for the transcription factors and verified that the purified proteins bind these motifs in vitro Furthermore, several direct target genes were coordinately and reciprocally regulated by Cir1 and HapX, suggesting that these transcription factors play conserved roles in the response to iron availability. In addition, biochemical analyses revealed that Cir1 and HapX are iron-containing proteins, implying that the regulatory networks of Cir1 and HapX may be influenced by the incorporation of iron into these proteins. Taken together, our identification of the genome-wide transcriptional networks provides a detailed understanding of the iron-related regulatory landscape, establishes a new connection between Cir1 and kinases that regulate capsule, and underpins genetic and biochemical analyses that reveal iron-sensing mechanisms for Cir1 and HapX in C. neoformans ., (Copyright © 2020 by the Genetics Society of America.)

Pathogens must compete with hosts to acquire sufficient iron for proliferation during pathogenesis. The pathogenic fungus Cryptococcus neoformans is capable of acquiring iron from heme, the most abundant source in vertebrate hosts, although the mechanisms of heme sensing and acquisition are not entirely understood. In this study, we adopted a chromosomally encoded heme sensor developed for Saccharomyces cerevisiae to examine cytosolic heme levels in C. neoformans using fluorescence microscopy, fluorimetry, and flow cytometry. We validated the responsiveness of the sensor upon treatment with exogenous hemin, during proliferation in macrophages, and in strains defective for endocytosis. We then used the sensor to show that vacuolar and mitochondrial dysregulation and oxidative stress reduced the labile heme pool in the cytosol. Importantly, the sensor provided a tool to further demonstrate that the drugs artemisinin and metformin have heme-related activities and the potential to be repurposed for antifungal therapy. Overall, this study provides insights into heme sensing by C. neoformans and establishes a powerful tool to further investigate mechanisms of heme-iron acquisition in the context of fungal pathogenesis. IMPORTANCE Invasive fungal diseases are increasing in frequency, and new drug targets and antifungal drugs are needed to bolster therapy. The mechanisms by which pathogens obtain critical nutrients such as iron from heme during host colonization represent a promising target for therapy. In this study, we employed a fluorescent heme sensor to investigate heme homeostasis in Cryptococcus neoformans We demonstrated that endocytosis is a key aspect of heme acquisition and that vacuolar and mitochondrial functions are important in regulating the pool of available heme in cells. Stress generated by oxidative conditions impacts the heme pool, as do the drugs artemisinin and metformin; these drugs have heme-related activities and are in clinical use for malaria and diabetes, respectively. Overall, our study provides insights into mechanisms of fungal heme acquisition and demonstrates the utility of the heme sensor for drug characterization in support of new therapies for fungal diseases., (Copyright © 2020 Bairwa et al.)

The opportunistic fungal pathogen Cryptococcus neoformans must adapt to the mammalian environment to establish an infection. Proteins facilitating adaptation to novel environments, such as chaperones, may be required for virulence. In this study, we identified a novel mitochondrial co-chaperone, Mrj1 ( m itochondrial r espiration J -domain protein 1 ), necessary for virulence in C. neoformans The mrj1Δ and J-domain-inactivated mutants had general growth defects at both routine laboratory and human body temperatures and were deficient in the major virulence factor of capsule elaboration. The latter phenotype was associated with cell wall changes and increased capsular polysaccharide shedding. Accordingly, the mrj1Δ mutant was avirulent in a murine model of cryptococcosis. Mrj1 has a mitochondrial localization and co-immunoprecipitated with Qcr2, a core component of complex III of the electron transport chain. The mrj1 mutants were deficient in mitochondrial functions, including growth on alternative carbon sources, growth without iron, and mitochondrial polarization. They were also insensitive to complex III inhibitors and hypersensitive to an alternative oxidase (AOX) inhibitor, suggesting that Mrj1 functions in respiration. In support of this conclusion, mrj1 mutants also had elevated basal oxygen consumption rates which were completely abolished by the addition of the AOX inhibitor, confirming that Mrj1 is required for mitochondrial respiration through complexes III and IV. Furthermore, inhibition of complex III phenocopied the capsule and cell wall defects of the mrj1 mutants. Taken together, these results indicate that Mrj1 is required for normal mitochondrial respiration, a key aspect of adaptation to the host environment and virulence. IMPORTANCE Cryptococcus neoformans is the causative agent of cryptococcal meningitis, a disease responsible for ∼15% of all HIV-related deaths. Unfortunately, development of antifungal drugs is challenging because potential targets are conserved between humans and C. neoformans In this context, we characterized a unique J-domain protein, Mrj1, which lacks orthologs in humans. We showed that Mrj1 was required for normal mitochondrial respiration and that mutants lacking Mrj1 were deficient in growth, capsule elaboration, and virulence. Furthermore, we were able to phenocopy the defects in growth and capsule elaboration by inhibiting respiration. This result suggests that the role of Mrj1 in mitochondrial function was responsible for the observed virulence defects and reinforces the importance of mitochondria to fungal pathogenesis. Mitochondria are difficult to target, as their function is also key to human cells; however, Mrj1 presents an opportunity to target a unique fungal protein required for mitochondrial function and virulence in C. neoformans ., (Copyright © 2020 Horianopoulos et al.)

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security., (Copyright © 2020 Fisher et al.)

Ustilago maydis is a model organism for the study of biotrophic plant-pathogen interactions. The sexual and pathogenic development of the fungus are tightly connected since fusion of compatible haploid sporidia is prerequisite for infection of the host plant, maize (Zea mays). After plant penetration, the unfolded protein response (UPR) is activated and required for biotrophic growth. The UPR is continuously active throughout all stages of pathogenic development in planta. However, since development of UPR deletion mutants stops directly after plant penetration, the role of an active UPR at later stages of development remained to be determined. Here, we established a gene expression system for U. maydis that uses endogenous, conditionally active promoters to either induce or repress expression of a gene of interest during different stages of plant infection. Integration of the expression constructs into the native genomic locus and removal of resistance cassettes were required to obtain a wild-type-like expression pattern. This indicates that genomic localization and chromatin structure are important for correct promoter activity and gene expression. By conditional expression of the central UPR regulator, Cib1, in U. maydis, we show that a functional UPR is required for continuous plant defence suppression after host infection and that U. maydis relies on a robust control system to prevent deleterious UPR hyperactivation., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Iron acquisition is essential for the proliferation of microorganisms, and human pathogens such as the fungus Cryptococcus neoformans must use sophisticated uptake mechanisms to overcome host iron sequestration. Iron is of particular interest for C. neoformans because its availability is an important cue for the elaboration of virulence factors. In fungi, extracellular iron is taken up through high affinity, low affinity, siderophore-mediated, and heme uptake pathways, and the details of these mechanisms are under active investigation in C. neoformans. Following uptake, iron is transported to intracellular organelles including mitochondria where it is used in heme biosynthesis and the synthesis of iron-sulfur (Fe-S) cluster precursors. One Fe-S cluster binding protein of note is the monothiol glutaredoxin Grx4 which has emerged as a master regulator of iron sensing in C. neoformans and other fungi through its influence on the expression of proteins for iron uptake or use. The activity of Grx4 likely occurs through interactions with Fe-S clusters and transcription factors known to control expression of the iron-related functions. Although the extent to which Grx4 controls the iron regulatory network is still being investigated in C. neoformans, it is remarkable that it also influences the expression of many genes encoding mitochondrial functions. Coupled with recent studies linking mitochondrial morphology and electron transport to virulence factor elaboration, there is an emerging appreciation of mitochondria as central players in cryptococcal disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nutrient sensing is critical for adaptation of fungi to environmental and host conditions. The conserved cAMP/PKA signaling pathway contributes to adaptation by sensing the availability of key nutrients such as glucose and directing changes in gene expression and metabolism. Interestingly, the cAMP/PKA pathway in fungal pathogens also influences the expression of virulence determinants in response to nutritional and host signals. For instance, protein kinase A (PKA) in the human pathogen Cryptococcus neoformans plays a central role in orchestrating phenotypic changes, such as capsule elaboration and melanin production, that directly impact disease development. In this review, we focus first on insights into the role of the cAMP/PKA pathway in nutrient sensing for the model yeast Saccharomyces cerevisiae to provide a foundation for understanding the pathway in C. neoformans . We then discuss key features of cAMP/PKA signaling in C. neoformans including new insights emerging from the analysis of transcriptional and proteomic changes in strains with altered PKA activity and expression. Finally, we highlight recent studies that connect the cAMP/PKA pathway to cell surface remodeling and the formation of titan cells.

Cryptococcus neoformans is a major fungal pathogen that infects immunocompromised people and causes life-threatening meningoencephalitis. C. neoformans does not occur in isolation either in the environment or in the human host, but is surrounded by other microorganisms. Bacteria are ubiquitously distributed in nature, including soil, and make up the dominant part of the human microbiota. Pioneering studies in the 1950s demonstrated antifungal activity of environmental bacteria against C. neoformans . However, the mechanisms and implications of these interactions remain largely unknown. Recently, interest in polymicrobial interaction studies has been reignited by the development of improved sequencing methodologies, and by the realization that such interactions may have a huge impact on ecology and human health. In this review, we summarize our current understanding of the interaction of bacteria with C. neoformans .

The acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungus Cryptococcus neoformans In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence in C. neoformans Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that a grx4 mutant lacking the GRX domain displayed iron-related phenotypes similar to those of a cir1 Δ mutant, including poor growth upon iron deprivation. Importantly, the grx4 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of the grx4 mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis. IMPORTANCE Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans , causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by which C. neoformans senses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability of C. neoformans to cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases., (Copyright © 2018 Attarian et al.)

Biotrophic fungal pathogens of plants must sense and adapt to the host environment to complete their life cycles. Recent transcriptome studies of the infection of maize by the biotrophic pathogen Ustilago maydis are providing molecular insights into an ordered program of changes in gene expression and the deployment of effectors as well as key features of nutrient acquisition. In particular, the transcriptome data provide a deeper appreciation of the complexity of the transcription factor network that controls the biotrophic program of invasion, proliferation, and sporulation. Additionally, transcriptome analysis during tumor formation, a key late stage in the life cycle, revealed features of the remodeling of host and pathogen metabolism that may support the formation of tremendous numbers of spores. Transcriptome studies are also appearing for other smut species during interactions with their hosts, thereby providing opportunities for comparative approaches to understand biotrophic adaptation., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Pathogenic microorganisms employ specialized virulence factors to cause disease. Biofilm formation and the production of a polysaccharide capsule are two important virulence factors in Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis. Here, we show that the bipolar disorder drug lithium inhibits formation of both virulence factors by a mechanism involving dysregulation of the ubiquitin/proteasome system. By using a chemical genetics approach and bioinformatic analyses, we describe the cellular landscape affected by lithium treatment. We demonstrate that lithium affects many different pathways in C. neoformans , including the cAMP/protein kinase A, inositol biosynthesis, and ubiquitin/proteasome pathways. By analyzing mutants with defects in the ubiquitin/proteasome system, we uncover a role for proteostasis in both capsule and biofilm formation. Moreover, we demonstrate an additive influence of lithium and the proteasome inhibitor bortezomib in inhibiting capsule production, thus establishing a link between lithium activity and the proteasome system. Finally, we show that the lithium-mimetic drug ebselen potently blocks capsule and biofilm formation, and has additive activity with lithium or bortezomib. In summary, our results illuminate the impact of lithium on C. neoformans, and link dysregulation of the proteasome to capsule and biofilm inhibition in this important fungal pathogen., Competing Interests: Conflict of interest: The authors declare that they have no conflicts of interest.

The process of autophagy is conserved among all eukaryotes from yeast to humans and is mainly responsible for bulk degradation of cellular contents and nutrient recycling during starvation. Autophagy has been suggested to play a role in the pathogenesis of the opportunistic human fungal pathogen Cryptococcus neoformans , potentially through a contribution to the export of virulence factors. In this study, we showed that deletion of each of the ATG1 , ATG7 , ATG8 , and ATG9 genes in C. neoformans leads to autophagy-related phenotypes, including impaired amino acid homeostasis under nitrogen starvation. In addition, the atg Δ mutants were hypersensitive to inhibition of the ubiquitin-proteasome system, a finding consistent with a role in amino acid homeostasis. Although each atg Δ mutant was not markedly impaired in virulence factor production in vitro , we found that all four ATG genes contribute to C. neoformans virulence in a murine inhalation model of cryptococcosis. Interestingly, these mutants displayed significant differences in their ability to promote disease development. A more detailed investigation of virulence for the atg1 Δ and atg8 Δ mutants revealed that both strains stimulated an exaggerated host immune response, which, in turn, contributed to disease severity. Overall, our results suggest that different ATG genes are involved in nonautophagic functions and contribute to C. neoformans virulence beyond their core functions in autophagy., (Copyright © 2018 American Society for Microbiology.)

The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and to antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence., Competing Interests: The authors have declared that no competing interests exist.

Iron-sulfur clusters (ISC) are indispensable cofactors for essential enzymes in various cellular processes. In the model yeast Saccharomyces cerevisiae, the precursor of ISCs is exported from mitochondria via a mitochondrial ABC transporter Atm1 and used for cytosolic and nuclear ISC protein assembly. Although iron homeostasis has been implicated in the virulence of the human fungal pathogen Cryptococcus neoformans, the key components of the ISC biosynthesis pathway need to be fully elucidated. In the current study, a homolog of S. cerevisiae Atm1 was identified in C. neoformans, and its function was characterized. We constructed C. neoformans mutants lacking ATM1 and found that deletion of ATM1 affected mitochondrial functions. Furthermore, we observed diminished activity of the cytosolic ISC-containing protein Leu1 and the heme-containing protein catalase in the atm1 mutant. These results suggested that Atm1 is required for the biosynthesis of ISCs in the cytoplasm as well as heme metabolism in C. neoformans. In addition, the atm1 mutants were avirulent in a murine model of cryptococcosis. Overall, our results demonstrated that Atm1 plays a critical role in iron metabolism and virulence for C. neoformans.