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

ABSTRACT 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.

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The Zinc Finger Protein Mig1 Regulates Mitochondrial Function and Azole Drug Susceptibility in the Pathogenic Fungus Cryptococcus neoformans

mSphere ◽

10.1128/msphere.00080-15 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 20

Author(s):

Mélissa Caza ◽

Guanggan Hu ◽

Michael Price ◽

John R. Perfect ◽

James W. Kronstad

Keyword(s):

Cryptococcus Neoformans ◽

Mitochondrial Function ◽

Drug Susceptibility ◽

Antifungal Drugs ◽

Antifungal Drug ◽

Oxygen Species ◽

Content Type ◽

New Targets ◽

Expression Of Genes ◽

Mitochondrial Functions

ABSTRACT Fungal pathogens of humans are difficult to treat, and there is a pressing need to identify new targets for antifungal drugs and to obtain a detailed understanding of fungal proliferation in vertebrate hosts. In this study, we examined the roles of the regulatory proteins Mig1 and HapX in mitochondrial function and antifungal drug susceptibility in the fungus Cryptococcus neoformans. This pathogen is a particular threat to the large population of individuals infected with human immunodeficiency virus (HIV). Our analysis revealed regulatory interactions between Mig1 and HapX, and a role for Mig1 in mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Importantly, loss of Mig1 increased susceptibility to the antifungal drug fluconazole, which is commonly used to treat cryptococcal disease. These studies highlight an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and tricarboxylic acid (TCA) cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions, and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 under low-iron conditions and Mig1 regulation of mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Consistently with these regulatory functions, a mig1Δ mutant had impaired growth on inhibitors of mitochondrial respiration and inducers of ROS. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole, thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. IMPORTANCE Fungal pathogens of humans are difficult to treat, and there is a pressing need to identify new targets for antifungal drugs and to obtain a detailed understanding of fungal proliferation in vertebrate hosts. In this study, we examined the roles of the regulatory proteins Mig1 and HapX in mitochondrial function and antifungal drug susceptibility in the fungus Cryptococcus neoformans. This pathogen is a particular threat to the large population of individuals infected with human immunodeficiency virus (HIV). Our analysis revealed regulatory interactions between Mig1 and HapX, and a role for Mig1 in mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Importantly, loss of Mig1 increased susceptibility to the antifungal drug fluconazole, which is commonly used to treat cryptococcal disease. These studies highlight an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs.

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

mBio ◽

10.1128/mbio.01127-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Linda C. Horianopoulos ◽

Guanggan Hu ◽

Mélissa Caza ◽

Kerstin Schmitt ◽

Peter Overby ◽

...

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

Mitochondrial Function ◽

Mitochondrial Respiration ◽

Antifungal Drugs ◽

Complex Iii ◽

Content Type ◽

Mitochondrial Functions ◽

J Domain ◽

Domain Protein

ABSTRACT 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. IMPORTANCE Cryptococcus 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.

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Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽

10.1128/msphere.00715-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Suresh Ambati ◽

Emma C. Ellis ◽

Jianfeng Lin ◽

Xiaorong Lin ◽

Zachary A. Lewis ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Effective Dose ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Antifungal Drugs ◽

Extracellular Matrices ◽

Content Type ◽

Order Of Magnitude ◽

Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

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Power Failure of Mitochondria and Oxidative Stress in Neurodegeneration and Its Computational Models

Antioxidants ◽

10.3390/antiox10020229 ◽

2021 ◽

Vol 10 (2) ◽

pp. 229

Author(s):

JunHyuk Woo ◽

Hyesun Cho ◽

YunHee Seol ◽

Soon Ho Kim ◽

Chanhyeok Park ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dysfunction ◽

Computational Models ◽

Neuronal Damage ◽

Living Organism ◽

The Body ◽

Mitochondrial Functions ◽

A Cell ◽

The Brain ◽

And Oxidative Stress

The brain needs more energy than other organs in the body. Mitochondria are the generator of vital power in the living organism. Not only do mitochondria sense signals from the outside of a cell, but they also orchestrate the cascade of subcellular events by supplying adenosine-5′-triphosphate (ATP), the biochemical energy. It is known that impaired mitochondrial function and oxidative stress contribute or lead to neuronal damage and degeneration of the brain. This mini-review focuses on addressing how mitochondrial dysfunction and oxidative stress are associated with the pathogenesis of neurodegenerative disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, and Parkinson’s disease. In addition, we discuss state-of-the-art computational models of mitochondrial functions in relation to oxidative stress and neurodegeneration. Together, a better understanding of brain disease-specific mitochondrial dysfunction and oxidative stress can pave the way to developing antioxidant therapeutic strategies to ameliorate neuronal activity and prevent neurodegeneration.

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Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

Nucleic Acids Research ◽

10.1093/nar/gkp509 ◽

2009 ◽

Vol 37 (14) ◽

pp. 4812-4825 ◽

Cited By ~ 40

Author(s):

José F. da Silva Neto ◽

Vânia S. Braz ◽

Valéria C. S. Italiani ◽

Marilis V. Marques

Keyword(s):

Oxidative Stress ◽

Iron Homeostasis ◽

Caulobacter Crescentus ◽

Oxidative Stress Defense ◽

And Oxidative Stress ◽

Stress Defense

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Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus

mBio ◽

10.1128/mbio.02735-21 ◽

2021 ◽

Author(s):

José P. Guirao-Abad ◽

Martin Weichert ◽

Ginés Luengo-Gil ◽

Sarah Sze Wah Wong ◽

Vishukumar Aimanianda ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Resistance ◽

Secretory Pathway ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Gene Encoding ◽

Content Type ◽

Downstream Target ◽

P Type ◽

Er Homeostasis

The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus .

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Dysfunction of Prohibitin 2 Results in Reduced Susceptibility to Multiple Antifungal Drugs via Activation of the Oxidative Stress-Responsive Transcription Factor Pap1 in Fission Yeast

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00860-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 1

Author(s):

Qiannan Liu ◽

Fan Yao ◽

Guanglie Jiang ◽

Min Xu ◽

Si Chen ◽

...

Keyword(s):

Oxidative Stress ◽

Drug Resistance ◽

Transcription Factor ◽

Fission Yeast ◽

Mitochondrial Protein ◽

Antifungal Drugs ◽

Antifungal Drug ◽

Gene Encoding ◽

Content Type ◽

Antifungal Drug Resistance

ABSTRACT The fight against resistance to antifungal drugs requires a better understanding of the underlying cellular mechanisms. In order to gain insight into the mechanisms leading to antifungal drug resistance, we performed a genetic screen on a model organism, Schizosaccharomyces pombe, to identify genes whose overexpression caused resistance to antifungal drugs, including clotrimazole and terbinafine. We identified the phb2+ gene, encoding a highly conserved mitochondrial protein, prohibitin (Phb2), as a novel determinant of reduced susceptibility to multiple antifungal drugs. Unexpectedly, deletion of the phb2+ gene also exhibited antifungal drug resistance. Overexpression of the phb2+ gene failed to cause drug resistance when the pap1+ gene, encoding an oxidative stress-responsive transcription factor, was deleted. Furthermore, pap1+ mRNA expression was significantly increased when the phb2+ gene was overexpressed or deleted. Importantly, either overexpression or deletion of the phb2+ gene stimulated the synthesis of NO and reactive oxygen species (ROS), as measured by the cell-permeant fluorescent NO probe DAF-FM DA (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) and the ROS probe DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate), respectively. Taken together, these results suggest that Phb2 dysfunction results in reduced susceptibility to multiple antifungal drugs by increasing NO and ROS synthesis due to dysfunctional mitochondria, thereby activating the transcription factor Pap1 in fission yeast.

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Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01698-19 ◽

2019 ◽

Vol 64 (3) ◽

Author(s):

Cody B. Bullock ◽

David S. McNabb ◽

Inés Pinto

Keyword(s):

Mechanism Of Action ◽

Fungal Infections ◽

Antifungal Drugs ◽

Content Type ◽

Histatin 5 ◽

Amino Acid Peptide ◽

Killing Activity ◽

Transport Chain ◽

Mitochondrial Functions ◽

Increased Sensitivity

ABSTRACT The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans. Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

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Host Carbon Dioxide Concentration Is an Independent Stress for Cryptococcus neoformans That Affects Virulence and Antifungal Susceptibility

mBio ◽

10.1128/mbio.01410-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 1

Author(s):

Damian J. Krysan ◽

Bing Zhai ◽

Sarah R. Beattie ◽

Kara M. Misel ◽

Melanie Wellington ◽

...

Keyword(s):

Carbon Dioxide ◽

Cryptococcus Neoformans ◽

Carbon Dioxide Concentration ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Mammalian Host ◽

Environmental Isolates ◽

Capsule Formation ◽

Content Type ◽

Virulence Traits

ABSTRACT The ability of Cryptococcus neoformans to cause disease in humans varies significantly among strains with highly related genotypes. In general, environmental isolates of pathogenic species such as Cryptococcus neoformans var. grubii have reduced virulence relative to clinical isolates, despite having no differences in the expression of the canonical virulence traits (high-temperature growth, melanization, and capsule formation). In this observation, we report that environmental isolates of C. neoformans tolerate host CO2 concentrations poorly compared to clinical isolates and that CO2 tolerance correlates well with the ability of the isolates to cause disease in mammals. Initial experiments also suggest that CO2 tolerance is particularly important for dissemination of C. neoformans from the lung to the brain. Furthermore, CO2 concentrations affect the susceptibility of both clinical and environmental C. neoformans isolates to the azole class of antifungal drugs, suggesting that antifungal testing in the presence of CO2 may improve the correlation between in vitro azole activity and patient outcome. IMPORTANCE A number of studies comparing either patient outcomes or model system virulence across large collections of Cryptococcus isolates have found significant heterogeneity in virulence even among strains with highly related genotypes. Because this heterogeneity cannot be explained by variations in the three well-characterized virulence traits (growth at host body temperature, melanization, and polysaccharide capsule formation), it has been widely proposed that additional C. neoformans virulence traits must exist. The natural niche of C. neoformans is in the environment, where the carbon dioxide concentration is very low (∼0.04%); in contrast, mammalian host tissue carbon dioxide concentrations are 125-fold higher (5%). We have found that the ability to grow in the presence of 5% carbon dioxide distinguishes low-virulence strains from high-virulence strains, even those with a similar genotype. Our findings suggest that carbon dioxide tolerance is a previously unrecognized virulence trait for C. neoformans.

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