Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2, which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug fluconazole caused increased vacuolar fragmentation. These conditions also commonly cause an increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly due to copper or iron limitation.

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Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00261-12 ◽

2012 ◽

Vol 11 (12) ◽

pp. 1482-1495 ◽

Cited By ~ 9

Author(s):

Dong-Hoon Yang ◽

Shinae Maeng ◽

Anna K. Strain ◽

Anna Floyd ◽

Kirsten Nielsen ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Stress Responses ◽

Fungal Pathogens ◽

Independent Manner ◽

Content Type ◽

Antifungal Drug Resistance ◽

Human Fungal Pathogen ◽

Assembly Factor ◽

Life Threatening ◽

Stress Related Genes

ABSTRACT Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans , which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans . Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78 . In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans , providing insight into a potential novel antifungal therapeutic target.

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Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming

Nature Communications ◽

10.1038/s41467-019-12907-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Amanda L. M. Bloom ◽

Richard M. Jin ◽

Jay Leipheimer ◽

Jonathan E. Bard ◽

Donald Yergeau ◽

...

Keyword(s):

Ribosomal Protein ◽

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Mrna Decay ◽

Wild Type Strain ◽

Core Body Temperature ◽

Temperature Adaptation ◽

Wild Type ◽

Transcriptomic Changes ◽

Core Body

Abstract A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans, is their ability to adapt to mammalian core body temperature. In C. neoformans, this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion.

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Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages

Infection and Immunity ◽

10.1128/iai.71.1.173-180.2003 ◽

2003 ◽

Vol 71 (1) ◽

pp. 173-180 ◽

Cited By ~ 179

Author(s):

Gary M. Cox ◽

Thomas S. Harrison ◽

Henry C. McDade ◽

Carlos P. Taborda ◽

Garrett Heinrich ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Superoxide Dismutase ◽

Cryptococcus Neoformans ◽

Fungal Infections ◽

Reactive Oxygen ◽

Oxygen Species ◽

Wild Type ◽

Pathogenic Yeast ◽

Antioxidant Defense Systems

ABSTRACT Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.

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Role of Alternative Oxidase Gene in Pathogenesis of Cryptococcus neoformans

Infection and Immunity ◽

10.1128/iai.71.10.5794-5802.2003 ◽

2003 ◽

Vol 71 (10) ◽

pp. 5794-5802 ◽

Cited By ~ 79

Author(s):

Shamima Akhter ◽

Henry C. McDade ◽

Jenifer M. Gorlach ◽

Garrett Heinrich ◽

Gary M. Cox ◽

...

Keyword(s):

Oxidative Stress ◽

Cryptococcus Neoformans ◽

Mutant Strain ◽

Alternative Oxidase ◽

Temperature Sensitive ◽

Wild Type ◽

Phagocytic Cells ◽

Pathogenic Yeast ◽

Oxidase Gene ◽

Oxidative Pathway

ABSTRACT We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37°C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.

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Skeletal muscle weakness due to deficiency of CuZn-superoxide dismutase is associated with loss of functional innervation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00093.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. R1400-R1407 ◽

Cited By ~ 49

Author(s):

Lisa M. Larkin ◽

Carol S. Davis ◽

Catrina Sims-Robinson ◽

Tatiana Y. Kostrominova ◽

Holly Van Remmen ◽

...

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Muscle Weakness ◽

Muscle Atrophy ◽

Muscle Stimulation ◽

Wild Type ◽

Functional Changes ◽

Fiber Number ◽

Cuzn Superoxide Dismutase

An association between oxidative stress and muscle atrophy and weakness in vivo is supported by elevated oxidative damage and accelerated loss of muscle mass and force with aging in CuZn-superoxide dismutase-deficient ( Sod1−/−) mice. The purpose was to determine the basis for low specific force (N/cm2) of gastrocnemius muscles in Sod1−/− mice and establish the extent to which structural and functional changes in muscles of Sod1−/− mice resemble those associated with normal aging. We tested the hypothesis that muscle weakness in Sod1−/− mice is due to functionally denervated fibers by comparing forces during nerve and direct muscle stimulation. No differences were observed for wild-type mice at any age in the forces generated in response to nerve and muscle stimulation. Nerve- and muscle-stimulated forces were also not different for 4-wk-old Sod1−/− mice, whereas, for 8- and 20-mo-old mice, forces during muscle stimulation were 16 and 30% greater, respectively, than those obtained using nerve stimulation. In addition to functional evidence of denervation with aging, fiber number was not different for Sod1−/− and wild-type mice at 4 wk, but 50% lower for Sod1−/− mice by 20 mo, and denervated motor end plates were prevalent in Sod1−/− mice at both 8 and 20 mo and in WT mice by 28 mo. The data suggest ongoing denervation in muscles of Sod1−/− mice that results in fiber loss and muscle atrophy. Moreover, the findings support using Sod1−/− mice to explore mechanistic links between oxidative stress and the progression of deficits in muscle structure and function.

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Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00058.2001 ◽

2002 ◽

Vol 282 (4) ◽

pp. L719-L726 ◽

Cited By ~ 80

Author(s):

Russell P. Bowler ◽

Mike Nicks ◽

Karrie Warnick ◽

James D. Crapo

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Pulmonary Fibrosis ◽

Lung Injury ◽

Immunohistochemical Staining ◽

Extracellular Superoxide Dismutase ◽

Wild Type ◽

Injured Lung ◽

Intracellular Oxidative Stress

Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 ± 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 ± 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 ± 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.

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Bladder function in mice with inducible smooth muscle-specific deletion of the manganese superoxide dismutase gene

AJP Cell Physiology ◽

10.1152/ajpcell.00046.2015 ◽

2015 ◽

Vol 309 (3) ◽

pp. C169-C178 ◽

Cited By ~ 4

Author(s):

Guiming Liu ◽

Rania A. Elrashidy ◽

Nan Xiao ◽

Michael Kavran ◽

Yexiang Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Smooth Muscle ◽

Manganese Superoxide Dismutase ◽

Bladder Function ◽

Antioxidant Systems ◽

Wild Type ◽

Manganese Superoxide ◽

Specific Deletion

Manganese superoxide dismutase (MnSOD) is considered a critical component of the antioxidant systems that protect against oxidative damage. We are interested in the role of oxidative stress in bladder detrusor smooth muscle (SM) in different disease states. In this study, we generated an inducible, SM-specific Sod2−/− mouse model to investigate the effects of MnSOD depletion on the function of the bladder. We crossbred floxed Sod2 ( Sod2lox/lox) mice with mice containing heterozygous knock-in of a gene encoding a tamoxifen-activated Cre recombinase in the SM22α promoter locus [SM-CreERT2(ki)Cre/+]. We obtained Sod2lox/lox,SM-CreERT2(ki)Cre/+ mice and injected 8-wk-old males with 4-hydroxytamoxifen to induce Cre-mediated excision of the floxed Sod2 allele. Twelve weeks later, SM-specific deletion of Sod2 and depletion of MnSOD were confirmed by polymerase chain reaction, immunoblotting, and immunohistochemistry. SM-specific Sod2−/− mice exhibited normal growth with no gross abnormalities. A significant increase in nitrotyrosine concentration was found in bladder SM tissue of SM-specific Sod2−/− mice compared with both wild-type mice and Sod2+/+, SM-CreERT2(ki)Cre/+ mice treated with 4-hydroxytamoxifen. Assessment of 24-h micturition in SM-specific Sod2−/− mice revealed significantly higher voiding frequency compared with both wild-type and SM-specific Cre controls. Conscious cystometry revealed significantly shorter intercontraction intervals and lower functional bladder capacity in SM-specific Sod2−/− mice compared with wild-type mice. This novel model can be used for exploring the mechanistic role of oxidative stress in organs rich in SM in different pathological conditions.

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Lysine Decarboxylase Expression by Vibrio vulnificus Is Induced by SoxR in Response to Superoxide Stress

Journal of Bacteriology ◽

10.1128/jb.01084-06 ◽

2006 ◽

Vol 188 (24) ◽

pp. 8586-8592 ◽

Cited By ~ 36

Author(s):

Ju-Sim Kim ◽

Sang Ho Choi ◽

Jeong K. Lee

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Promoter Region ◽

Methyl Viologen ◽

Vibrio Vulnificus ◽

External Medium ◽

Gene Dosage ◽

Acid Stress ◽

Lysine Decarboxylase ◽

Wild Type

ABSTRACT Lysine decarboxylase expression by Vibrio vulnificus, which is up-regulated by CadC in response to acid stress, is also induced by SoxR in response to superoxide stress. SoxR binds to the promoter region of the cadBA operon, coding for a lysine-cadaverine antiporter (CadB) and a lysine decarboxylase (CadA). The induction of cadBA transcription by SoxR is independent of CadC. Cadaverine, which neutralizes the external medium, also appears to scavenge superoxide radicals, since increasing cellular cadaverine by elevating the gene dosage of cadBA significantly diminished the induction of Mn-containing superoxide dismutase under methyl viologen-induced oxidative stress. Consistently, a lack of cadaverine caused by mutation in cadA resulted in low tolerance to oxidative stress compared with that of the wild type.

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Random T-DNA Mutagenesis Identifies a Cu/Zn Superoxide Dismutase Gene as a Virulence Factor of Sclerotinia sclerotiorum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-12-0177-r ◽

2013 ◽

Vol 26 (4) ◽

pp. 431-441 ◽

Cited By ~ 29

Author(s):

Liangsheng Xu ◽

Weidong Chen

Keyword(s):

Oxidative Stress ◽

Growth Rate ◽

Superoxide Dismutase ◽

Sclerotinia Sclerotiorum ◽

Virulence Factor ◽

Wild Type ◽

Expression Levels ◽

Sclerotial Formation ◽

Increased Sensitivity ◽

And Oxidative Stress

Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed growth rate, sclerotial formation, and oxalate production similar to that of the wild type. The mutation was due to a single T-DNA insertion at 212 bp downstream of the Cu/Zn superoxide dismutase (SOD) gene (SsSOD1, SS1G_00699). Expression levels of SsSOD1 were significantly increased under oxidative stresses or during plant infection in the wild-type strain but could not be detected in the mutant. SsSOD1 functionally complemented the Cu/Zn SOD gene in a Δsod1 Saccharomyces cerevisiae mutant. The SOD mutant had increased sensitivity to heavy metal toxicity and oxidative stress in culture and reduced ability to detoxify superoxide in infected leaves. The mutant also had reduced expression levels of other known pathogenicity genes such as endo-polygalacturanases sspg1 and sspg3. The functions of SsSOD1 were further confirmed by SsSOD1-deletion mutation. Like the AMT insertion mutant, the SsSOD1-deletion mutant exhibited normal growth rate, sclerotial formation, oxalate production, increased sensitivity to metal and oxidative stress, and reduced virulence. These results suggest that SsSOD1, while not being required for saprophytic growth and completion of the life cycle, plays critical roles in detoxification of reactive oxygen species during host–pathogen interactions and is an important virulence factor of Sclerotinia sclerotiorum.

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Cryptococcus neoformans Mitochondrial Superoxide Dismutase: an Essential Link between Antioxidant Function and High-Temperature Growth

Eukaryotic Cell ◽

10.1128/ec.4.1.46-54.2005 ◽

2005 ◽

Vol 4 (1) ◽

pp. 46-54 ◽

Cited By ~ 67

Author(s):

Steven S. Giles ◽

Ines Batinić-Haberle ◽

John R. Perfect ◽

Gary M. Cox

Keyword(s):

Oxidative Stress ◽

Superoxide Dismutase ◽

Cryptococcus Neoformans ◽

Manganese Superoxide Dismutase ◽

Antioxidant Defense ◽

Elevated Temperatures ◽

Antioxidant Defense System ◽

Defense System ◽

Poor Growth ◽

Manganese Superoxide

ABSTRACT Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Δ mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Δ mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 μM) at 30°C, but not at 37°C. Furthermore, sod2Δ mutant cells that were incubated for 24 h at 37°C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25°C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.

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