Genetic Analysis of Candida albicans Filamentation by the Iron Chelator BPS Reveals a Role for a Conserved Kinase-WD40 Protein Pair.

Candida albicans is a major human pathogenic fungus that is distinguished by its capability to switch from a yeast to a hyphal morphology under different conditions. Here, we analyze the cellular effects of high concentrations of the iron chelator bathophenanthroline disulfonate (BPS). BPS inhibits cellular growth by withholding iron, but when iron chelation is overcome by the addition of hemoglobin as an iron source, the cells resume growth as hyphae. The BPS hyphal induction pathway was characterized by identifying the hyphal-specific transcription factors that it requires and by a forward genetic screen for mutants that fail to form hyphae in BPS using a transposon library generated in a haploid strain. Among the mutants identified are the DYRK1-like kinase Yak1 and Orf19.384, a homolog of the DYRK1-associated protein WDR68/DCAF7. Orf19.384 nuclear localization depends on Yak1, similar to their mammalian counterparts. We identified the hyphal suppressor transcription factor Sfl1 as a candidate target of Yak1-Orf19.384 and show that Sfl1 modification is similarly affected in the yak1 and orf19.384 mutant strains. These results suggest that DYRK1/Yak1 and WDR68/Orf19.384 represent a conserved protein pair that regulates cell differentiation from fungi to animals.

Ferric reductase-related proteins mediate fungal heme acquisition.

Heme can serve as iron source in many environments, including the iron-poor animal host environment. The fungal pathobiont Candida albicans expresses a family of extracellular CFEM hemophores that capture heme from host proteins and transfer it across the cell wall to the cell membrane, to be endocytosed and utilized as heme or iron source. Here, we identified Frp1 and Frp2, two ferric reductase (FRE)-related proteins that lack an extracellular N-terminal substrate-binding domain, as being required for hemoglobin heme utilization and for sensitivity to toxic heme analogs. Frp1 and Frp2 redistribute to the plasma membrane in the presence of hemin, consistent with a direct role in heme trafficking. Expression of Frp1 with the CFEM hemophore Pga7 can promote heme utilization in Saccharomyces cerevisiae as well, confirming the functional interaction between these proteins. Sequence and structure comparison reveals that the CFEM hemophores are related to the FRE substrate-binding domain that is missing in Frp1/2. We conclude that Frp1/2 and the CFEM hemophores form a functional complex that evolved from FREs to enable extracellular heme uptake.

Hosts and disease-causing fungi are often locked into a battle over resources. The host will attempt to withhold molecules that the fungus needs to survive, while the pathogen will try to find alternative routes to obtain them. Candida albicans, for example, can go after the atoms of iron embedded in the proteins of the organism it infects. To do so it releases molecules known as hemophores, which scavenge the iron-containing heme molecule that equips oxygen-carrying proteins in the blood. Once captured, the heme is carried across the wall that protects C. albicans from the environment and brought to the membrane of the cell. It is then taken in and trafficked inside vesicles to its destination. However, the identity of the molecular actors which help to bridge the internal and external segments of the heme journey remain unclear. Previous studies have shown that the hemophore Pga7 is involved, but this protein is attached to the outside of the cell membrane, where it cannot directly interact with the import machinery. Roy et al. set out to discover this missing link. Examining the genomes of fungal species related to C. albicans highlighted two membrane proteins, Frp1 and Frp2, which could participate in heme uptake. Protein sequence comparison revealed that Frp1 and Frp2 were closely related to ferric reductases, a group of membrane enzymes which can chemically alter extracellular iron prior to uptake. Deleting the genes for Frp1 and Frp2 rendered C. albicans cells incapable of taking in heme. Conversely, a fungal species which cannot normally uptake heme could efficiently internalise these complexes when artificially equipped with Frp1 and Pga7, suggesting that the two proteins work closely together. Finally, protein structure comparisons highlighted that an extracellular domain present in ferric reductases but absent in Frp1 and Frp2 is, in fact, related to Pga7 and other hemophores. This implies that the iron and heme uptake systems may share a common evolutionary origin. Overall, the work by Roy et al. reveals a new family of proteins which allow disease-causing fungi to steal iron from their hosts. This knowledge may be useful to design better anti-fungal treatments.

Regulation of heme utilization and homeostasis in Candida albicans.

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble and anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor related to S. cerevisiae HAP1. In the presence of hemin in the medium, C. albicans HAP1 is induced, the Hap1 protein is stabilized and Hap1-GFP localizes to the nucleus. In the hap1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Hap1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Hap1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by C. albicans Hap1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph.

Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans.

Candida albicans is a major fungal pathogen that can utilise hemin and haemoglobin as iron sources in the iron-scarce host environment. While C. albicans is a heme prototroph, we show here that it can also efficiently utilise external heme as a cellular heme source. Using genetically encoded ratiometric fluorescent heme sensors, we show that heme extracted from haemoglobin and free hemin enter the cells with different kinetics. Heme supplied as haemoglobin is taken up via the Common in Fungal Extracellular Membrane (CFEM) hemophore cascade, and reaches the cytoplasm over several hours, whereas entry of free hemin via CFEM-dependent and independent pathways is much faster, less than an hour. To prevent an influx of extracellular heme from reaching toxic levels in the cytoplasm, the cells deploy Hmx1, a heme oxygenase. Hmx1 was previously suggested to be involved in utilisation of haemoglobin and hemin as iron sources, but we find that it is primarily required to prevent heme toxicity. Taken together, the combination of novel heme sensors with genetic analysis revealed new details of the fungal mechanisms of heme import and homeostasis, necessary to balance the uses of heme as essential cofactor and potential iron source against its toxicity.

Human Serum Albumin Facilitates Heme-Iron Utilization by Fungi.

A large portion of biological iron is found in the form of an iron-protoporphyrin IX complex, or heme. In the human host environment, which is exceptionally poor in free iron, heme iron, particularly from hemoglobin, constitutes a major source of iron for invading microbial pathogens. Several fungi were shown to utilize free heme, and Candida albicans, a major opportunistic pathogen, is able both to capture free heme and to extract heme from hemoglobin using a network of extracellular hemophores. Human serum albumin (HSA) is the most abundant host heme-scavenging protein. Tight binding of heme by HSA restricts its toxic chemical reactivity and could diminish its availability as an iron source for pathogenic microbes. We found, however, that rather than inhibiting heme utilization, HSA greatly increases availability of heme as an iron source for C. albicans and other fungi. In contrast, hemopexin, a low-abundance but high-affinity heme-scavenging serum protein, does inhibit heme utilization by C. albicans However, inhibition by hemopexin is mitigated in the presence of HSA. Utilization of albumin-bound heme requires the same hemophore cascade as that which mediates hemoglobin-iron utilization. Accordingly, we found that the C. albicans hemophores are able to extract heme bound to HSA in vitro Since many common drugs are known to bind to HSA, we tested whether they could interfere with heme-iron utilization. We show that utilization of albumin-bound heme by C. albicans can be inhibited by the anti-inflammatory drugs naproxen and salicylic acid.IMPORTANCE Heme constitutes a major iron source for microorganisms and particularly for pathogenic microbes; to overcome the iron scarcity in the animal host, many pathogenic bacteria and fungi have developed systems to extract and take up heme from host proteins such as hemoglobin. Microbial heme uptake mechanisms are usually studied using growth media containing free heme or hemoglobin as a sole iron source. However, the animal host contains heme-scavenging proteins that could prevent this uptake. In the human host in particular, the most abundant serum heme-binding protein is albumin. Surprisingly, however, we found that in the case of fungi of the Candida species family, albumin promoted rather than prevented heme utilization. Albumin thus constitutes a human-specific factor that can affect heme-iron utilization and could serve as target for preventing heme-iron utilization by fungal pathogens. As a proof of principle, we identify two drugs that can inhibit albumin-stimulated heme utilization.

Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis.

Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8 were only detected in the hyphal cells. Genetic analysis of these sites revealed allele-specific effects on growth or hyphae formation at 42 °C. Colony morphology, which is normally wrinkled or crenellated at 37 °C, reverted to smooth in several mutants, but this colony morphology phenotype was unrelated to cellular morphology. Two mutants exhibited a mild increase in sensitivity to the cell wall-active compounds caspofungin and calcofluor white. We suggest that this analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence. The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client proteins, and that modification of these sites can play cellular regulatory roles, consistent with the "chaperone code" hypothesis.

A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge.

We investigated whether long-lived weight-reduced αMUPA mice differ from their wild types in postnatal body composition and leptin level, and whether these differences are affected by maternal-borne factors. Newborn αMUPA and wild type mice had similar body weight and composition up to the third postnatal week, after which αMUPA mice maintained lower body weight due to lower fat-free mass. Both strains showed a surge in leptin levels at the second postnatal week, initiating earlier in αMUPA mice, rising higher and lasting longer than in the wild types, mainly in females. Leptin level in dams' serum and breast milk, and in their pup's stomach content were also higher in αMUPA than in the WT during the surge peak. Leptin surge preceded the strain divergence in body weight, and was associated with an age-dependent decrease in the leptin:fat mass ratio-suggesting that postnatal sex and strain differences in leptin ontogeny are strongly influenced by processes independent of fat mass, such as production and secretion, and possibly outside fat tissues. Dam removal elevated corticosterone level in female pups from both strains similarly, yet mitigated the leptin surge only in αMUPA-eliminating the strain differences in leptin levels. Overall, our results indicate that αMUPA's postnatal leptin surge is more pronounced than in the wild type, more sensitive to maternal deprivation, less related to pup's total adiposity, and is associated with a lower post-weaning fat-free mass. These strain-related postnatal differences may be related to αMUPA's higher milk-borne leptin levels. Thus, our results support the use of αMUPA mice in future studies aimed to explore the relationship between maternal (i.e. milk-borne) factors, postnatal leptin levels, and post-weaning body composition and energy homeostasis.

Regulation of the Candida albicans Hypha-Inducing Transcription Factor Ume6 by the CDK1 Cyclins Cln3 and Hgc1.

The ability to switch between proliferation as yeast cells and development into hyphae is a hallmark of Candida albicans. The switch to hyphal morphogenesis depends on external inducing conditions, but its efficiency is augmented in stationary-phase cells. Ume6, a transcription factor that is itself transcriptionally induced under hypha-promoting conditions, is both necessary and sufficient for hyphal morphogenesis. We found that Ume6 is regulated posttranslationally by the cell cycle kinase Cdc28/Cdk1, which reduces Ume6 activity via different mechanisms using different cyclins. Together with the cyclin Hgc1, Cdk1 promotes degradation of Ume6 via the SCFCDC4 ubiquitin ligase. Since HGC1 is a key transcriptional target of Ume6, this results in a negative-feedback loop between Hgc1 and Ume6. In addition, we found that Cln3, a G1 cyclin that is essential for cell cycle progression and yeast proliferation, suppresses hyphal morphogenesis and that Cln3 suppresses Ume6 activity both in the heterologous Saccharomyces cerevisiae system and in C. albicans itself. This activity of Cln3 may provide the basis for the antagonistic relationship between yeast proliferation and hyphal development in C. albicans. IMPORTANCE The yeast to hypha (mold) morphogenetic switch of Candida albicans plays a role in its virulence and constitutes a diagnostic trait for this organism, the most prevalent systemic fungal pathogen in industrialized countries. It has long been known that hyphae are most efficiently induced from stationary cultures. Here, a molecular basis for this observation is provided. The G1 cyclin Cln3, an essential promoter of yeast proliferation, was found to suppress hyphal induction. Suppression of hyphal induction is achieved by inhibition of the activity of the central activator of hyphal morphogenesis, the transcription factor Ume6. Thus, levels of Cln3 control the switch between proliferation of C. albicans as individual yeast cells and development into extended hyphae, a switch that may preface the proliferation/differentiation switch in multicellular organisms.

Structural basis of haem-iron acquisition by fungal pathogens.

Pathogenic microorganisms must cope with extremely low free-iron concentrations in the host's tissues. Some fungal pathogens rely on secreted haemophores that belong to the Common in Fungal Extracellular Membrane (CFEM) protein family, to extract haem from haemoglobin and to transfer it to the cell's interior, where it can serve as a source of iron. Here we report the first three-dimensional structure of a CFEM protein, the haemophore Csa2 secreted by Candida albicans. The CFEM domain adopts a novel helical-basket fold that consists of six α-helices, and is uniquely stabilized by four disulfide bonds formed by its eight signature cysteines. The planar haem molecule is bound between a flat hydrophobic platform located on top of the helical basket and a peripheral N-terminal 'handle' extension. Exceptionally, an aspartic residue serves as the CFEM axial ligand, and so confers coordination of Fe3+ haem, but not of Fe2+ haem. Histidine substitution mutants of this conserved Asp acquired Fe2+ haem binding and retained the capacity to extract haem from haemoglobin. However, His-substituted CFEM proteins were not functional in vivo and showed disturbed haem exchange in vitro, which suggests a role for the oxidation-state-specific Asp coordination in haem acquisition by CFEM proteins.

Long-Lived αMUPA Mice Show Reduced Sexual Dimorphism in Lifespan, and in Energy and Circadian Homeostasis-Related Parameters.

Female αMUPA (alpha murine urokinase-like plasminogen activator) transgenic mice show increased lifespan, reduced body weight and food intake, and high-amplitude circadian rhythms with an endogenous period length (tau) of 24h, versus their wild types (WT) showing a 23.7-h tau. Our goal was to characterize αMUPA and WT male mice, and their in-strain sexual dimorphism, and to further understand the mechanisms underlying αMUPA's longevity. Male αMUPA mice showed increased lifespan, reduced body weight and food intake, and aligned endogenous rhythm with a tau of 24.0h versus a tau <24h in WT. However, no differences were found when intake was corrected for metabolic mass in male αMUPA mice. αMUPA's sexual dimorphism was damped or lacking in all studied traits, while WTs were sexually dimorphic, concluding that αMUPA's transgene overrides sex-dependent mechanisms involved in lifespan and in energy and circadian homeostasis. As enhanced resonance between tau and external circadian cycle correlates with increased lifespan and reduced body weight in other species, including humans, αMUPA's 24-h tau could contribute to their longevity. Focusing future research on the mechanistic interconnections between energy homeostasis, circadian homeostasis, sexual dimorphism, and aging, using αMUPA mice, may reveal mechanisms promoting reduced body weight and increased lifespan.