The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

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.

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

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 blocked upon loss of 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. Additionally, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially blocked 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.

Heme sensing and trafficking in fungi.

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.

Proteasome inhibition as a therapeutic target for the fungal pathogen Cryptococcus neoformans.

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.

Metals and the cell surface of Cryptococcus neoformans.

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.

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen.

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.

Vam6/Vps39/TRAP1-domain proteins influence vacuolar morphology, iron acquisition and virulence in Cryptococcus neoformans.

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.

The monothiol glutaredoxin Grx4 influences thermotolerance, cell wall integrity, and Mpk1 signaling in Cryptococcus neoformans.

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.

Dnj1 Promotes Virulence in Cryptococcus neoformans by Maintaining Robust Endoplasmic Reticulum Homeostasis Under Temperature Stress.

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.

Involvement of Mrs3/4 in Mitochondrial Iron Transport and Metabolism in Cryptococcus neoformans.

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.

The Novel J-Domain Protein Mrj1 Is Required for Mitochondrial Respiration and Virulence in Cryptococcus neoformans.

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 (mitochondrial respiration 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.IMPORTANCECryptococcus 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.

Role of clathrin-mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans.

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life-threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non-iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin-mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin-containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.

The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus 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 cir1Δ 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.

The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans.

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.

ATG Genes Influence the Virulence of Cryptococcus neoformans through Contributions beyond Core Autophagy Functions.

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 atgΔ mutants were hypersensitive to inhibition of the ubiquitin-proteasome system, a finding consistent with a role in amino acid homeostasis. Although each atgΔ 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 atg1Δ and atg8Δ 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.

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans.

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

A P4-ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans.

The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of the cell division control protein 50 (Cdc50) family of the noncatalytic subunit of type IV P-type ATPases (flippases) that establish phospholipid asymmetry in membranes and function in vesicle-mediated trafficking. We found that a cdc50 mutant in C. neoformans was defective for survival in macrophages, attenuated for virulence in mice and impaired in iron acquisition. The mutant also showed increased sensitivity to drugs associated with phospholipid metabolism (cinnamycin and miltefosine), the antifungal drug fluconazole and curcumin, an iron chelator that accumulates in the endoplasmic reticulum. Cdc50 is expected to function with catalytic subunits of flippases, and we previously documented the involvement of the flippase aminophospholipid translocases (Apt1) in virulence factor delivery. A comparison of phenotypes with mutants defective in genes encoding candidate flippases (designated APT1, APT2, APT3, and APT4) revealed similarities primarily between cdc50 and apt1 suggesting a potential functional interaction. Overall, these results highlight the importance of membrane composition and homeostasis for the ability of C. neoformans to cause disease.

Fungal Glycolipid Hydrolase Inhibitors and Their Effect on Cryptococcus neoformans.

Pathogenic fungi kill an estimated 1.3 million people each year. This number is predicted to rise as drug resistance spreads, thus antifungal drugs with novel modes of action are urgently required. Fungal endoglycoceramidase-related proteins 1 and 2 (EGCrP-1 and -2), which hydrolyse glucosylceramide and ergosteryl ß-glucoside, respectively, are important for fungal cell growth and have been identified as potential targets for drug development. A library of iminosugar derivatives was screened against EGCrP-1 and -2, and a number of competitive inhibitors with nanomolar affinities were identified. In addition, a mechanism-based inhibitor was shown to form a covalent derivative with EGCrP-2. Nine of the inhibitors were evaluated against Cryptococcus neoformans. Several showed growth inhibitory activity, but only against a C. neoformans strain lacking the outer fungal polysaccharide capsule; this implies that penetration into the cell is a significant handicap for these inhibitors. Pro-drug versions of these inhibitors could address this issue.

The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis.