A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans.

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.

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.

A Transcriptional Regulatory Map of Iron Homeostasis Reveals a New Control Circuit for Capsule Formation in Cryptococcus neoformans.

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.

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 mitochondrial ABC transporter Atm1 plays a role in iron metabolism and virulence in the human fungal pathogen Cryptococcus neoformans.

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.

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.

The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans.

Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in the ESCRT-II and III complexes caused reduced capsule attachment, impaired growth on haem and resistance to non-iron metalloprotoporphyrins. The ESCRT mutants shared several phenotypes with a mutant lacking the pH-response regulator Rim101, and in other fungi, the ESCRT machinery is known to activate Rim101 via proteolytic cleavage. We therefore expressed a truncated and activated version of Rim101 in the ESCRT mutants and found that this allele restored capsule formation but not growth on haem, thus suggesting a Rim101-independent contribution to haem uptake. We also demonstrated that the ESCRT machinery acts downstream of the cAMP/protein kinase A pathway to influence capsule elaboration. Defects in the ESCRT components also attenuated virulence in macrophage survival assays and a mouse model of cryptococcosis to a greater extent than reported for loss of Rim101. Overall, these results indicate that the ESCRT complexes function in capsule elaboration, haem uptake and virulence via Rim101-dependent and independent mechanisms.

Leu1 plays a role in iron metabolism and is required for virulence in Cryptococcus neoformans.

Amino acid biosynthetic pathways that are absent in mammals are considered an attractive target for antifungal therapy. Leucine biosynthesis is one such target pathway, consisting of a five-step conversion process starting from the valine precursor 2-keto-isovalerate. Isopropylmalate dehydrogenase (Leu1) is an Fe-S cluster protein that is required for leucine biosynthesis in the model fungus Saccharomyces cerevisiae. The human pathogenic fungus Cryptococcus neoformans possesses an ortholog of S. cerevisiae Leu1, and our previous transcriptome data showed that the expression of LEU1 is regulated by iron availability. In this study, we characterized the role of Leu1 in iron homeostasis and the virulence of C. neoformans. We found that deletion of LEU1 caused leucine auxotrophy and that Leu1 may play a role in the mitochondrial-cytoplasmic Fe-S cluster balance. Whereas cytoplasmic Fe-S protein levels were not affected, mitochondrial Fe-S proteins were up-regulated in the leu1 mutant, suggesting that Leu1 mainly influences mitochondrial iron metabolism. The leu1 mutant also displayed increased sensitivity to oxidative stress and cell wall/membrane disrupting agents, which may have been caused by mitochondrial dysfunction. Furthermore, the leu1 mutant was deficient in capsule formation and showed attenuated virulence in a mouse inhalation model of cryptococcosis. Overall, our results indicate that Leu1 plays a role in iron metabolism and is required for virulence in C. neoformans.

Iron acquisition in the human fungal pathogen Cryptococcus neoformans.

Iron sequestration by the vertebrate host is considered an efficient defense mechanism against pathogenic microbes. However, this mechanism, so called nutritional immunity, is often overcome by the iron acquisition systems that have evolved in microbial pathogens. Numerous studies have been carried out to identify the key components of these systems and to understand their underlying mechanisms, including recent investigations in the basidiomycete fungal pathogen Cryptococcus neoformans. Iron acquisition is essential for the survival and pathogenesis of this fungus within vertebrate hosts. Growing evidence suggests that the fungus is able to utilize several different iron sources available in the host, and that the intracellular or extracellular localization of the pathogen influences its iron acquisition strategy. Herein, we review current findings on the components and regulatory elements of the iron acquisition systems in C. neoformans.

A human fungal pathogen Cryptococcus neoformans expresses three distinct iron permease homologs.

Iron plays a key role in host-pathogen interactions. Microbial pathogens require iron for survival and virulence, whereas mammalian hosts sequester and withhold iron as a means of nutritional immunity. We previously identified two paralogous genes, CFT1 and CFT2, which encode homologs of a fungal iron permease, Cft1 and Cft2, respectively, in the human fungal pathogen Cryptococcus neoformans. Cft1 was shown to play a role in the high-affinity reductive iron uptake system, and was required for transferrin utilization and full virulence in mammalian hosts. However, no role of Cft2 has been suggested yet. Here, we identified the third gene, CFT3, that produces an additional fungal iron permease homolog in C. neoformans, and we also generated the cft3 mutant for functional characterization. We aimed to reveal distinct functions of Cft1, Cft2 and Cft3 by analyzing phenotypes of the mutants lacking CFT1, CFT2 and CFT3, respectively. The endogenous promoter of CFT1, CFT2 and CFT3 was replaced with the inducible GAL7 promoter in the wildtype strain or in the cft1 mutant for gain-of-function analysis. Using these strains, we were able to find that CFT2 is required for growth in low-iron conditions in the absence of CFT1 and that overexpression of CFT2 compensates for deficiency of the cft1 mutant in iron uptake and various cellular stress conditions. However, unlike CFT2, no clear phenotypic characteristic of the cft3 mutant and the strain overexpressing CFT3 was observed. Overall, our data suggested a redundant role of Cft2 in the high-affinity iron uptake and stress responses in C. neoformans.

A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs.

The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD(+)/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.