Characterization of a selective, iron-chelating antifungal compound that disrupts fungal metabolism and synergizes with fluconazole.

Fungal infections are a growing global health concern due to the limited number of available antifungal therapies as well as the emergence of fungi that are resistant to first-line antimicrobials, particularly azoles and echinocandins. Development of novel, selective antifungal therapies is challenging due to similarities between fungal and mammalian cells. An attractive source of potential antifungal treatments is provided by ecological niches co-inhabited by bacteria, fungi, and multicellular organisms, where complex relationships between multiple organisms have resulted in evolution of a wide variety of selective antimicrobials. Here, we characterized several analogs of one such natural compound, collismycin A. We show that NR-6226C has antifungal activity against several pathogenic Candida species, including C. albicans and C. glabrata, whereas it only has little toxicity against mammalian cells. Mechanistically, NR-6226C selectively chelates iron, which is a limiting factor for pathogenic fungi during infection. As a result, NR-6226C treatment causes severe mitochondrial dysfunction, leading to formation of reactive oxygen species, metabolic reprogramming, and a severe reduction in ATP levels. Using an in vivo model for fungal infections, we show that NR-6226C significantly increases survival of Candida-infected Galleria mellonella larvae. Finally, our data indicate that NR-6226C synergizes strongly with fluconazole in inhibition of C. albicans. Taken together, NR-6226C is a promising antifungal compound that acts by chelating iron and disrupting mitochondrial functions.IMPORTANCEDrug-resistant fungal infections are an emerging global threat, and pan-resistance to current antifungal therapies is an increasing problem. Clearly, there is a need for new antifungal drugs. In this study, we characterized a novel antifungal agent, the collismycin analog NR-6226C. NR-6226C has a favorable toxicity profile for human cells, which is essential for further clinical development. We unraveled the mechanism of action of NR-6226C and found that it disrupts iron homeostasis and thereby depletes fungal cells of energy. Importantly, NR-6226C strongly potentiates the antifungal activity of fluconazole, thereby providing inroads for combination therapy that may reduce or prevent azole resistance. Thus, NR-6226C is a promising compound for further development into antifungal treatment.

Iron drives anabolic metabolism through active histone demethylation and mTORC1.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

Iron Regulatory Mechanisms in Saccharomyces cerevisiae.

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in many cellular processes. However, excess iron can damage cells since it promotes the generation of reactive oxygen species. The budding yeast Saccharomyces cerevisiae has been used as a model organism to study the adaptation of eukaryotic cells to changes in iron availability. Upon iron deficiency, yeast utilizes two transcription factors, Aft1 and Aft2, to activate the expression of a set of genes known as the iron regulon, which are implicated in iron uptake, recycling and mobilization. Moreover, Aft1 and Aft2 activate the expression of Cth2, an mRNA-binding protein that limits the expression of genes encoding for iron-containing proteins or that participate in iron-using processes. Cth2 contributes to prioritize iron utilization in particular pathways over other highly iron-consuming and non-essential processes including mitochondrial respiration. Recent studies have revealed that iron deficiency also alters many other metabolic routes including amino acid and lipid synthesis, the mitochondrial retrograde response, transcription, translation and deoxyribonucleotide synthesis; and activates the DNA damage and general stress responses. At high iron levels, the yeast Yap5, Msn2, and Msn4 transcription factors activate the expression of a vacuolar iron importer called Ccc1, which is the most important high-iron protecting factor devoted to detoxify excess cytosolic iron that is stored into the vacuole for its mobilization upon scarcity. The complete sequencing and annotation of many yeast genomes is starting to unveil the diversity and evolution of the iron homeostasis network in this species.

The yeast Aft1 transcription factor activates ribonucleotide reductase catalytic subunit RNR1 in response to iron deficiency.

Eukaryotic ribonucleotide reductases are iron-dependent enzymes that catalyze the rate-limiting step in the de novo synthesis of deoxyribonucleotides. Multiple mechanisms regulate the activity of ribonucleotide reductases in response to genotoxic stresses and iron deficiency. Upon iron starvation, the Saccharomyces cerevisiae Aft1 transcription factor specifically binds to iron-responsive cis elements within the promoter of a group of genes, known as the iron regulon, activating their transcription. Members of the iron regulon participate in iron acquisition, mobilization and recycling, and trigger a genome-wide metabolic remodeling of iron-dependent pathways. Here, we describe a mechanism that optimizes the activity of yeast ribonucleotide reductase when iron is scarce. We demonstrate that Aft1 and the DNA-binding protein Ixr1 enhance the expression of the gene encoding for its catalytic subunit, RNR1, in response to iron limitation, leading to an increase in both mRNA and protein levels. By mutagenesis of the Aft1-binding sites within RNR1 promoter, we conclude that RNR1 activation by iron depletion is important for Rnr1 protein and deoxyribonucleotide synthesis. Remarkably, Aft1 also activates the expression of IXR1 upon iron scarcity through an iron-responsive element located within its promoter. These results provide a novel mechanism for the direct activation of ribonucleotide reductase function by the iron-regulated Aft1 transcription factor.

Global translational repression induced by iron deficiency in yeast depends on the Gcn2/eIF2α pathway.

Iron is an essential element for all eukaryotic organisms because it participates as a redox active cofactor in a wide range of biological processes, including protein synthesis. Translation is probably the most energy consuming process in cells. Therefore, one of the initial responses of eukaryotic cells to stress or nutrient limitation is the arrest of mRNA translation. In first instance, the budding yeast Saccharomyces cerevisiae responds to iron deficiency by activating iron acquisition and remodeling cellular metabolism in order to prioritize essential over non-essential iron-dependent processes. We have determined that, despite a global decrease in transcription, mRNA translation is actively maintained during a short-term exposure to iron scarcity. However, a more severe iron deficiency condition induces a global repression of translation. Our results indicate that the Gcn2-eIF2α pathway limits general translation at its initiation step during iron deficiency. This bulk translational inhibition depends on the uncharged tRNA sensing Gcn1-Gcn20 complex. The involvement of the Gcn2-eIF2α pathway in the response to iron deficiency highlights its central role in the eukaryotic response to stress or nutritional deprivation, which is conserved from yeast to mammals.

A genome-wide transcriptional study reveals that iron deficiency inhibits the yeast TORC1 pathway.

Iron is an essential micronutrient that participates as a cofactor in a broad range of metabolic processes including mitochondrial respiration, DNA replication, protein translation and lipid biosynthesis. Adaptation to iron deficiency requires the global reorganization of cellular metabolism directed to optimize iron utilization. The budding yeast Saccharomyces cerevisiae has been widely used to characterize the responses of eukaryotic microorganisms to iron depletion. In this report, we used a genomic approach to investigate the contribution of transcription rates to the modulation of mRNA levels during adaptation of yeast cells to iron starvation. We reveal that a decrease in the activity of all RNA polymerases contributes to the down-regulation of many mRNAs, tRNAs and rRNAs. Opposite to the general expression pattern, many genes including components of the iron deficiency response, the mitochondrial retrograde pathway and the general stress response display a remarkable increase in both transcription rates and mRNA levels upon iron limitation, whereas genes encoding ribosomal proteins or implicated in ribosome biogenesis exhibit a pronounced fall. This expression profile is consistent with an activation of the environmental stress response. The phosphorylation stage of multiple regulatory factors strongly suggests that the conserved nutrient signaling pathway TORC1 is inhibited during the progress of iron deficiency. These results suggest an intricate crosstalk between iron metabolism and the TORC1 pathway that should be considered in many disorders.

Dissecting mRNA decay and translation inhibition during iron deficiency.

Iron participates as a vital cofactor in multiple metabolic pathways. Despite its abundance, iron bioavailability is highly restricted in aerobic and alkaline environments. Therefore, living organisms have evolved multiple adaptive mechanisms to respond to iron scarcity. These strategies include a global remodeling of iron metabolism directed to optimize iron utilization. In the baker's yeast Saccharomyces cerevisiae, this metabolic reorganization is accomplished to a large extent by an mRNA-binding protein called Cth2. Yeast Cth2 belongs to a conserved family of tandem zinc finger containing proteins that specifically bind to transcripts with AU-rich elements and promote their turnover. A recent study has revealed that Cth2 also inhibits the translation of its target mRNAs (Ramos-Alonso et al., PLoS Genet 14:e1007476, https://doi.org/10.1371/journal.pgen.1007476 , 2018). Interestingly, the mammalian Cth2 ortholog known as tristetraprolin (aka TTP/TIS11/ZFP36), which is also implicated in controlling iron metabolism, promotes the decay and prevents the translation of its regulated transcripts. These observations open the possibility to study the relative contribution of altering mRNA stability and translation to the physiological adaptation to iron deficiency, the function played by the different domains within the mRNA-binding protein, and the potential factors implicated in coordinating both post-transcriptional events.

Molecular strategies to increase yeast iron accumulation and resistance.

All eukaryotic organisms rely on iron as an essential micronutrient for life because it participates as a redox-active cofactor in multiple biological processes. However, excess iron can generate reactive oxygen species that damage cellular macromolecules. The low solubility of ferric iron under physiological conditions increases the prevalence of iron deficiency anemia. A common strategy to treat iron deficiency consists of dietary iron supplementation. The baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, but also as a feed supplement. In response to iron deficiency, the yeast Aft1 transcription factor activates cellular iron acquisition. However, when constitutively active, Aft1 inhibits growth probably due to iron toxicity. In this report, we have studied the consequences of using hyperactive AFT1 alleles, including AFT1-1UP, to increase yeast iron accumulation. We first characterized the iron sensitivity of cells expressing different constitutively active AFT1 alleles. We rescued the high iron sensitivity conferred by the AFT1 alleles by deleting the sphingolipid signaling kinase YPK1. We observed that the deletion of YPK1 exerts different effects on iron accumulation depending on the AFT1 allele and the environmental iron. Moreover, we determined that the impairment of the high-affinity iron transport system partially rescues the high iron toxicity of AFT1-1UP-expressing cells. Finally, we observed that AFT1-1UP inhibits oxygen consumption through activation of the RNA-binding protein Cth2. Deletion of CTH2 partially rescues the AFT1-1UP negative respiratory effect. Collectively, these results contribute to understand how the Aft1 transcription factor functions and the multiple consequences derived from its constitutive activation.

mRNA-binding protein tristetraprolin is essential for cardiac response to iron deficiency by regulating mitochondrial function.

Cells respond to iron deficiency by activating iron-regulatory proteins to increase cellular iron uptake and availability. However, it is not clear how cells adapt to conditions when cellular iron uptake does not fully match iron demand. Here, we show that the mRNA-binding protein tristetraprolin (TTP) is induced by iron deficiency and degrades mRNAs of mitochondrial Fe/S-cluster-containing proteins, specifically Ndufs1 in complex I and Uqcrfs1 in complex III, to match the decrease in Fe/S-cluster availability. In the absence of TTP, Uqcrfs1 levels are not decreased in iron deficiency, resulting in nonfunctional complex III, electron leakage, and oxidative damage. Mice with deletion of Ttp display cardiac dysfunction with iron deficiency, demonstrating that TTP is necessary for maintaining cardiac function in the setting of low cellular iron. Altogether, our results describe a pathway that is activated in iron deficiency to regulate mitochondrial function to match the availability of Fe/S clusters.

Yeast Cth2 protein represses the translation of ARE-containing mRNAs in response to iron deficiency.

In response to iron deficiency, the budding yeast Saccharomyces cerevisiae undergoes a metabolic remodeling in order to optimize iron utilization. The tandem zinc finger (TZF)-containing protein Cth2 plays a critical role in this adaptation by binding and promoting the degradation of multiple mRNAs that contain AU-rich elements (AREs). Here, we demonstrate that Cth2 also functions as a translational repressor of its target mRNAs. By complementary approaches, we demonstrate that Cth2 protein inhibits the translation of SDH4, which encodes a subunit of succinate dehydrogenase, and CTH2 mRNAs in response to iron depletion. Both the AREs within SDH4 and CTH2 transcripts, and the Cth2 TZF are essential for translational repression. We show that the role played by Cth2 as a negative translational regulator extends to other mRNA targets such as WTM1, CCP1 and HEM15. A structure-function analysis of Cth2 protein suggests that the Cth2 amino-terminal domain (NTD) is important for both mRNA turnover and translation inhibition, while its carboxy-terminal domain (CTD) only participates in the regulation of translation, but is dispensable for mRNA degradation. Finally, we demonstrate that the Cth2 CTD is physiologically relevant for adaptation to iron deficiency.

The elemental role of iron in DNA synthesis and repair.

Iron is an essential redox element that functions as a cofactor in many metabolic pathways. Critical enzymes in DNA metabolism, including multiple DNA repair enzymes (helicases, nucleases, glycosylases, demethylases) and ribonucleotide reductase, use iron as an indispensable cofactor to function. Recent striking results have revealed that the catalytic subunit of DNA polymerases also contains conserved cysteine-rich motifs that bind iron-sulfur (Fe/S) clusters that are essential for the formation of stable and active complexes. In line with this, mitochondrial and cytoplasmic defects in Fe/S cluster biogenesis and insertion into the nuclear iron-requiring enzymes involved in DNA synthesis and repair lead to DNA damage and genome instability. Recent studies have shown that yeast cells possess multi-layered mechanisms that regulate the ribonucleotide reductase function in response to fluctuations in iron bioavailability to maintain optimal deoxyribonucleotide concentrations. Finally, a fascinating DNA charge transport model indicates how the redox active Fe/S centers present in DNA repair machinery components are critical for detecting and repairing DNA mismatches along the genome by long-range charge transfers through double-stranded DNA. These unexpected connections between iron and DNA replication and repair have to be considered to properly understand cancer, aging and other DNA-related diseases.