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.

Cryptococcus neoformans can utilize ferritin as an iron source.

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

Coordinated regulation of iron metabolism in Cryptococcus neoformans by GATA and CCAAT transcription factors: connections with virulence.

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

An Accelerated Wound-Healing Surgical Suture Engineered with an Extracellular Matrix.

A suture is a ubiquitous medical device to hold wounded tissues together and support the healing process after surgery. Surgical sutures, having incomplete biocompatibility, often cause unwanted infections or serious secondary trauma to soft or fragile tissue. In this research, UV/ozone (UVO) irradiation or polystyrene sulfonate acid (PSS) dip-coating is used to achieve a fibronectin (FN)-coated absorbable suture system, in which the negatively charged moieties produced on the suture cause fibronectin to change from a soluble plasma form into a fibrous form, mimicking the actions of cellular fibronectin upon binding. The fibrous fibronectin coated on the suture can be exploited as an engineered interface to improve cellular migration and adhesion in the region around the wounded tissue while preventing the binding of infectious bacteria, thereby facilitating wound healing. Furthermore, the FN-coated suture is found to be associated with a lower friction between the suture and the wounded tissue, thus minimizing the occurrence of secondary wounds during surgery. It is believed that this surface modification can be universally applied to most kinds of sutures currently in use, implying that it may be a novel way to develop a highly effective and safer suture system for clinical applications.

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 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.

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.

In Vitro Anti-Malassezia Activity of Castanea crenata Shell and Oil-Soluble Glycyrrhiza Extracts.

BACKGROUND: A new shampoo with anti-Malassezia properties obtained from various plants is required to provide seborrheic dermatitis patients with a wider range of treatment options. OBJECTIVE: The aim of this study was to obtain in vitro susceptibility profiles of Malassezia restricta and M. globosa, the most important pathogenic organisms in the development of seborrheic dermatitis, to the plant extracts used in commercial anti-dandruff shampoos. METHODS: Minimal inhibitory concentrations (MICs) were determined for eight candidate plant extracts and two plant-derived natural products diluted with Leeming and Notman medium to final concentrations of 0.016 to 1 mg/ml. RESULTS: Castanea crenata shell, Camellia sinensis leaf, and oil-soluble Glycyrrhiza extracts presented relatively low MIC values (≤0.5 mg/ml) against both strains. The C. crenata shell and oil-soluble Glycyrrhiza extracts demonstrated especially high anti-Malassezia activity, suggesting their potential use in the treatment of seborrheic dermatitis. The extracts also showed fungistatic activity against other common facultative pathogenic yeasts, Cryptococcus and Candida. CONCLUSION: C. crenata shell and oil-soluble Glycyrrhiza extracts could potentially be used as active ingredients in anti-seborrheic and anti-dandruff shampoo formulations. They could be helpful for repeated treatments and regular prophylaxis of scalp seborrheic dermatitis.

Spontaneous Peripheral Ameloblastic Odontoma in a Male Sprague-Dawley Rat.

Peripheral ameloblastic odontoma is a rare variant of odontogenic tumor occurring in the extraosseous region. The present report describes a spontaneous tumor in male Sprague-Dawley (SD) rats. The clinically confirmed nodule in the right mandibular region was first observed when the rat was 42 weeks and remained until the terminal sacrifice date when the animal was 48 weeks of age. At necropsy, a well demarcated nodule, approximately 2.5 × 2.0 × 2.0 cm, protruded from the ventral area of the right mandible. The nodule was not attached to mandibular bone and was not continuous with the normal teeth. Histopathologically, the tumor was characterized by the simultaneous occurrence of an ameloblastomatous component and composite odontoma-like elements within the same tumor. The epithelial portion formed islands or cords resembling the follicle or plexiform pattern typical of ameloblastoma and was surrounded by mesenchymal tissue. Formation of eosinophilic and basophilic hard tissue matrix (dentin and enamel) resembling odontoma was observed in the center of the tumor. Mitotic figures were rare, and areas of cystic degeneration were present. Immunohistochemically, the epithelial component was positive for cytokeratin AE1/AE3 (CK AE1/AE3), and the mesenchymal component and odontoblast-like cells were positive for vimentin, in the same manner as in normal teeth. On the basis of these findings, the tumor was diagnosed as a peripheral ameloblastic odontoma in an extraosseous mandibular region in a SD rat. In the present study, we report the uncommon spontaneous peripheral ameloblastic odontoma in the SD rat. We also discuss here the morphological characteristics, origin, histochemical, and immunohistochemical features for the diagnosis of this tumor.

Mael is essential for cancer cell survival and tumorigenesis through protection of genetic integrity.

Germ line-specific genes are activated in somatic cells during tumorigenesis, and are accordingly referred to as cancer germline genes. Such genes that act on piRNA (Piwi-interacting RNA) processing play an important role in the progression of cancer cells. Here, we show that the spermatogenic transposon silencer maelstrom (Mael), a piRNA-processing factor, is required for malignant transformation and survival of cancer cells. A specific Mael isoform was distinctively overexpressed in diverse human cancer cell lines and its depletion resulted in cancer-specific cell death, characterized by apoptosis and senescence, accompanied by an increase in reactive oxygen-species and DNA damage. These biochemical changes and death phenotypes induced by Mael depletion were dependent on ATM. Interestingly Mael was essential for Myc/Ras-induced transformation, and its overexpression inhibited Ras-induced senescence. In addition, Mael repressed retrotransposon activity in cancer cells. These results suggest that Mael depletion induces ATM-dependent DNA damage, consequently leading to cell death specifically in cancer cells. Moreover, Mael possesses oncogenic potential that can protect against genetic instability.

Termisoflavones A-C, Isoflavonoid Glycosides from Termite-Associated Streptomyces sp. RB1.

Three new isoflavonoid glycosides, termisoflavones A-C (1-3), and eight isoflavonoids (4-11) were isolated from termite-associated Streptomyces sp. RB1 recovered from the cuticle of the South African termite, Macrotermes natalensis. The structures of new compounds were determined by spectroscopic methods including 1D and 2D NMR and HR-MS analysis, as well as comparison of their NMR data with those of related isoflavonoid glycoside derivatives. The absolute configurations of the sugar moieties were clarified by chemical reactions. None of the isolates (1-11) displayed antifungal or antimicrobial activities (MICs > 100 µg/mL), whereas compounds 6 and 11 ameliorated cisplatin-induced kidney cell damage to 80% of the control value at a cisplatin dose of 25 µM.

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.

p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2.

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

The cAMP/protein kinase A signaling pathway in pathogenic basidiomycete fungi: Connections with iron homeostasis.

A number of pathogenic species of basidiomycete fungi are either life-threatening pathogens of humans or major economic pests for crop production. Sensing the host is a key aspect of pathogen proliferation during disease, and signal transduction pathways are critically important for detecting environmental conditions and facilitating adaptation. This review focuses on the contributions of the cAMP/protein kinase A (PKA) signaling pathway in Cryptococcus neoformans, a species that causes meningitis in humans, and Ustilago maydis, a model phytopathogen that causes a smut disease on maize. Environmental sensing by the cAMP/PKA pathway regulates the production of key virulence traits in C. neoformans including the polysaccharide capsule and melanin. For U. maydis, the pathway controls the dimorphic transition from budding growth to the filamentous cell type required for proliferation in plant tissue. We discuss recent advances in identifying new components of the cAMP/PKA pathway in these pathogens and highlight an emerging theme that pathway signaling influences iron acquisition.