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1.
Planta Med ; 90(7-08): 588-594, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38843798

RESUMEN

Antimicrobial photodynamic therapy (aPDT) is an evolving treatment strategy against human pathogenic microbes such as the Candida species, including the emerging pathogen C. auris. Using a modified EUCAST protocol, the light-enhanced antifungal activity of the natural compound parietin was explored. The photoactivity was evaluated against three separate strains of five yeasts, and its molecular mode of action was analysed via several techniques, i.e., cellular uptake, reactive electrophilic species (RES), and singlet oxygen yield. Under experimental conditions (λ = 428 nm, H = 30 J/cm2, PI = 30 min), microbial growth was inhibited by more than 90% at parietin concentrations as low as c = 0.156 mg/L (0.55 µM) for C. tropicalis and Cryptococcus neoformans, c = 0.313 mg/L (1.10 µM) for C. auris, c = 0.625 mg/L (2.20 µM) for C. glabrata, and c = 1.250 mg/L (4.40 µM) for C. albicans. Mode-of-action analysis demonstrated fungicidal activity. Parietin targets the cell membrane and induces cell death via ROS-mediated lipid peroxidation after light irradiation. In summary, parietin exhibits light-enhanced fungicidal activity against all Candida species tested (including C. auris) and Cryptococcus neoformans, covering three of the four critical threats on the WHO's most recent fungal priority list.


Asunto(s)
Antifúngicos , Cryptococcus neoformans , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/efectos de la radiación , Candida auris/efectos de los fármacos , Luz , Candida/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Fotoquimioterapia/métodos , Antraquinonas/farmacología , Fármacos Fotosensibilizantes/farmacología
2.
Environ Microbiol ; 21(8): 2613-2628, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30724440

RESUMEN

The pathogenic fungus Cryptococcus neoformans produces melanin within its cell wall for infection and resistance against external stresses such as exposure to UV, temperature fluctuations and reactive oxygen species. It has been reported that melanin may also protect cells from ionizing radiation damage, against which C. neoformans is extremely resistant. This has tagged melanin as a potential radioprotective biomaterial. Here, we report the effect of melanin on the transcriptomic response of C. neoformans to gamma radiation. We did not observe a substantial protective effect of melanin against gamma radiation, and the general gene expression patterns in irradiated cells were independent of the presence of melanin. However, melanization itself dramatically altered the C. neoformans transcriptome, primarily by repressing genes involved in respiration and cell growth. We suggest that, in addition to providing a physical and chemical barrier against external stresses, melanin production alters the transcriptional landscape of C. neoformans with the result of increased resistance to uncertain environmental conditions. This observation demonstrates the importance of the melanization process in understanding the stress response of C. neoformans and for understanding fungal physiology.


Asunto(s)
Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/efectos de la radiación , Rayos gamma , Melaninas/metabolismo , Pared Celular/metabolismo , Criptococosis , Cryptococcus neoformans/efectos de los fármacos , Perfilación de la Expresión Génica , Tolerancia a Radiación
3.
Environ Microbiol ; 19(4): 1612-1624, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28127878

RESUMEN

Melanin is a ubiquitous pigment with unique physicochemical properties. The resistance of melanized fungi to cosmic and terrestrial ionizing radiation suggests that melanin also plays a pivotal role in radioprotection. In this study, we compared the effects of densely-ionizing deuterons and sparsely-ionizing X-rays on two microscopic fungi capable of melanogenesis. We utilized the fast-growing pathogenic basiodiomycete forming an induced DOPA-melanin, Cryptococcus neoformans (CN); and the slow-growing environmental rock-inhabiting ascomycete synthesizing a constitutive DHN-melanin, Cryomyces antarcticus (CA); melanized and non-melanized counterparts were compared. CA was more resistant to deuterons than CN, and similar resistance was observed for X-rays. Melanin afforded protection against high-dose (1.5 kGy) deuterons for both CN and CA (p-values < 10-4 ). For X-rays (0.3 kGy), melanin protected CA (p-values < 10-4 ) and probably CN. Deuterons increased XTT activity in melanized strains of both species, while the activity in non-melanized cells remained stable or decreased. For ATP levels the reverse occurred: it decreased in melanized strains, but not in non-melanized ones, after deuteron exposure. For both XTT and ATP, which reflect the metabolic activity of the cells, larger and more statistically-significant differences as a function of melanization status occurred in CN. Our data show, for the first time, that melanin protected both fast-growing and slow-growing fungi from high doses of deuterons under physiological conditions. These observations may give clues for creating melanin-based radioprotectors.


Asunto(s)
Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/efectos de la radiación , Melaninas/farmacología , Protectores contra Radiación/farmacología , Rayos X
4.
PLoS Genet ; 9(9): e1003769, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24039606

RESUMEN

The pathogenic fungus Cryptococcus neoformans uses the Bwc1-Bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. How the complex controls these functions is unclear. Here, we identify and characterize a gene in Cryptococcus, UVE1, whose mutation leads to a UV hypersensitive phenotype. The homologous gene in fission yeast Schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the UVDE-dependent excision repair (UVER) pathway. C. neoformans UVE1 complements a S. pombe uvde knockout strain. UVE1 is photoregulated in a Bwc1-dependent manner in Cryptococcus, and in Neurospora crassa and Phycomyces blakesleeanus that are species that represent two other major lineages in the fungi. Overexpression of UVE1 in bwc1 mutants rescues their UV sensitivity phenotype and gel mobility shift experiments show binding of Bwc2 to the UVE1 promoter, indicating that UVE1 is a direct downstream target for the Bwc1-Bwc2 complex. Uve1-GFP fusions localize to the mitochondria. Repair of UV-induced damage to the mitochondria is delayed in the uve1 mutant strain. Thus, in C. neoformans UVE1 is a key gene regulated in response to light that is responsible for tolerance to UV stress for protection of the mitochondrial genome.


Asunto(s)
Cryptococcus neoformans/efectos de los fármacos , Endodesoxirribonucleasas/genética , Genoma Mitocondrial/genética , Hipersensibilidad/genética , Proteínas de Schizosaccharomyces pombe/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/efectos de la radiación , Daño del ADN/efectos de la radiación , ADN de Hongos/genética , ADN de Hongos/efectos de la radiación , Endodesoxirribonucleasas/metabolismo , Técnicas de Inactivación de Genes , Genoma Mitocondrial/efectos de la radiación , Mutación , Neurospora crassa/genética , Neurospora crassa/efectos de la radiación , Phycomyces/genética , Phycomyces/efectos de la radiación , Schizosaccharomyces/genética , Schizosaccharomyces/efectos de la radiación , Proteínas de Schizosaccharomyces pombe/metabolismo , Rayos Ultravioleta
5.
Cell Microbiol ; 16(4): 473-81, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24438186

RESUMEN

The pathogenic fungi Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans are an increasing cause of human mortality, especially in immunocompromised populations. During colonization and adaptation to various host environments, these fungi undergo morphogenetic alterations that allow for survival within the host. One key environmental cue driving morphological changes is external temperature. The Hsp90 chaperone protein provides one mechanism to link temperature with the signalling cascades that regulate morphogenesis, fungal development and virulence. Candida albicans is a model system for understanding the connections between morphogenesis and Hsp90. Due to the high degree of conservation in Hsp90, many of the connections in C. albicans may be extrapolated to other fungal pathogens or parasites. Examining the role of Hsp90 during development and morphogenesis in these three major fungal pathogens may provide insight into key aspects of adaptation to the host, leading to additional avenues for therapy.


Asunto(s)
Candida albicans/fisiología , Candida albicans/efectos de la radiación , Regulación Fúngica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Aspergillus fumigatus/citología , Aspergillus fumigatus/patogenicidad , Aspergillus fumigatus/fisiología , Aspergillus fumigatus/efectos de la radiación , Candida albicans/citología , Candida albicans/patogenicidad , Cryptococcus neoformans/citología , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/fisiología , Cryptococcus neoformans/efectos de la radiación , Temperatura , Virulencia
6.
Mol Microbiol ; 89(1): 65-83, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23659661

RESUMEN

The pathogenic fungus Cryptococcus neoformans must overcome multiple stressors to cause disease in its human host. In this study, we report that C. neoformans rapidly and transiently repressed ribosomal protein (RP) transcripts during a transition from 30°C to host temperature. This repression was accompanied by accelerated mRNA degradation mediated by the major deadenylase, Ccr4, and influenced by the dissociable RNA polymerase II subunit, Rpb4. Destabilization and deadenylation of RP transcripts were impaired in an rpb4Δ mutant, suggesting that Rpb4 may be involved in host temperature-induced Ccr4-mediated decay. Accelerated decay of ER stress transcripts 1 h following a shift to host temperature was also impaired in the rpb4Δ mutant. In response to host temperature, Rpb4 moved from the nucleus to the cytoplasm, supporting a role for Rpb4 in coupling transcription and degradation. The PKH signalling pathway was implicated as a regulator of accelerated degradation of the RP transcripts, but not of the ER stress transcripts, revealing a further level of specificity. When transcription and degradation were uncoupled by deletion of Rpb4, growth at host temperature was impaired and virulence was attenuated. These data suggest that mRNA synthesis and decay are coupled in C. neoformans via Rpb4, and this tight coordination promotes host-temperature adaptation and pathogenicity.


Asunto(s)
Cryptococcus neoformans/fisiología , Cryptococcus neoformans/efectos de la radiación , Regulación Fúngica de la Expresión Génica , Estabilidad del ARN , ARN Mensajero/biosíntesis , Estrés Fisiológico , Cryptococcus neoformans/genética , Proteínas Fúngicas/metabolismo , Transducción de Señal , Temperatura
7.
PLoS Pathog ; 6(6): e1000945, 2010 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-20585557

RESUMEN

The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.


Asunto(s)
Criptococosis/microbiología , Criptococosis/patología , Cryptococcus neoformans/patogenicidad , Gigantismo/microbiología , Enfermedades Pulmonares Fúngicas/microbiología , Animales , Proliferación Celular , Criptococosis/inmunología , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/efectos de la radiación , ADN de Hongos/genética , Femenino , Técnica del Anticuerpo Fluorescente , Rayos gamma , Gigantismo/inmunología , Enfermedades Pulmonares Fúngicas/inmunología , Enfermedades Pulmonares Fúngicas/patología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estrés Oxidativo , Fagosomas/inmunología , Fagosomas/microbiología , Fagosomas/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Environ Microbiol Rep ; 14(4): 679-685, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35852045

RESUMEN

As human activity in space continues to increase, understanding how biological assets respond to spaceflight conditions is becoming more important. Spaceflight conditions include exposure to ionizing radiation, microgravity, spacecraft vibrations and hypervelocity; all of which can affect the viability of biological organisms. Previous studies have shown that melanin-producing fungi are capable of surviving the vacuum of space and Mars-simulated conditions in Low Earth Orbit. This survival has been associated in part with the protective effects of melanin, but a comparison of fungal viability in the presence or absence of melanin following spaceflight has never been tested. In this study, we evaluated the protective effects of melanin by comparing the viability of melanized and non-melanized clones of Cryptococcus neoformans yeasts following a roundtrip to the International Space Station. Yeast colonies were placed inside two MixStix silicone tubes; one stayed on Earth and the other was transported inside for 29 days before returning to Earth. Post-flight analysis based on colony-forming unit numbers shows that melanized yeast viability was 50% higher than non-melanized yeasts, while no difference was observed between the Earth-bound control samples. The results suggest that fungal melanin could increase the lifespan of biological assets in space.


Asunto(s)
Cryptococcus neoformans , Vuelo Espacial , Cryptococcus neoformans/efectos de la radiación , Humanos , Melaninas , Saccharomyces cerevisiae
9.
Microbiology (Reading) ; 156(Pt 8): 2393-2407, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20488877

RESUMEN

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.


Asunto(s)
Cryptococcus neoformans/enzimología , Ferroquelatasa/metabolismo , Proteínas Fúngicas/metabolismo , Luz , Cryptococcus neoformans/genética , Cryptococcus neoformans/efectos de la radiación , Ferroquelatasa/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Prueba de Complementación Genética , Mitocondrias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Phycomyces/genética , ARN de Hongos/genética , Saccharomyces cerevisiae/genética
10.
Med Mycol ; 48(7): 949-58, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20429770

RESUMEN

In addition to widespread use in reducing the symptoms of colds and flu, Echinacea is traditionally employed to treat fungal and bacterial infections. However, to date the mechanism of antimicrobial activity of Echinacea extracts remains unclear. We utilized a set of ∼4,600 viable gene deletion mutants of Saccharomyces cerevisiae to identify mutations that increase sensitivity to Echinacea. Thus, a set of chemical-genetic profiles for 16 different Echinacea treatments was generated, from which a consensus set of 23 Echinacea-sensitive mutants was identified. Of the 23 mutants, only 16 have a reported function. Ten of these 16 are involved in cell wall integrity/structure suggesting that a target for Echinacea is the fungal cell wall. Follow-up analyses revealed an increase in sonication-associated cell death in the yeasts S. cerevisiae and Cryptococcus neoformans after Echinacea extract treatments. Furthermore, fluorescence microscopy showed that Echinacea-treated S. cerevisiae was significantly more prone to cell wall damage than non-treated cells. This study further demonstrates the potential of gene deletion arrays to understand natural product antifungal mode of action and provides compelling evidence that the fungal cell wall is a target of Echinacea extracts and may thus explain the utility of this phytomedicine in treating mycoses.


Asunto(s)
Antifúngicos/farmacología , Pared Celular/efectos de los fármacos , Cryptococcus neoformans/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Echinacea/química , Extractos Vegetales/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Pared Celular/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/efectos de la radiación , Eliminación de Gen , Genes Fúngicos/fisiología , Pruebas de Sensibilidad Microbiana , Mutación Puntual , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de la radiación , Rayos Ultravioleta
11.
Antimicrob Agents Chemother ; 53(4): 1679-82, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19139285

RESUMEN

We investigated the utility of radioimmunotherapy (RIT) in the treatment of experimental cryptococcal infection with high-inoculum and the possibility of RIT treatment selecting for fungal cells with radiation-resistant phenotypes. RIT reduced mortality in high-burden infections, and we found no evidence for the development of radiation-resistant cells.


Asunto(s)
Criptococosis/radioterapia , Cryptococcus neoformans/efectos de la radiación , Radioinmunoterapia , Animales , Encéfalo/microbiología , Encéfalo/patología , Criptococosis/microbiología , Criptococosis/patología , Pulmón/microbiología , Pulmón/patología , Ratones , Tolerancia a Radiación
12.
Fungal Genet Biol ; 46(1): 42-54, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18996495

RESUMEN

Blue light regulates diverse physiological and developmental processes in fungi. Our prior studies demonstrated that the evolutionally conserved Cwc1 and Cwc2 proteins mediate the blue light-inhibited sexual filamentation in Cryptococcus neoformans. To characterize the putative domains of the Cwc1 and Cwc2 proteins, we generated partially deleted versions of these genes under the GPD1 promoter and examined their effects. The results confirmed that LOV and PAS domains are essential for the function of the Cwc1 protein, and the PAS domain and zinc finger DNA-binding motif are also crucial for the Cwc2 protein. To further understand how light inhibits filamentous growth, a genome wide mutant screening was conducted to identify genes important for this process. Mutants which suppressed the light-dependent CWC1 overexpression phenotype and restored mating filamentation were identified. In the one with fully restored filamentation, the T-DNA was found to disrupt the expression of the CWC2 gene. Additionally, a mediator component, the SSN8 gene, known to involve in transcriptional regulation was also identified. Our results demonstrate that Cwc1 and Cwc2 are two central regulators of the C. neoformans photoresponses and the roles of other components identified in the screen are under investigation.


Asunto(s)
Cryptococcus neoformans/fisiología , Genes Fúngicos , Luz , Mutación/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/efectos de la radiación , ADN Bacteriano/genética , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Genes Supresores , Mutagénesis Insercional , Fenotipo , Estructura Terciaria de Proteína/fisiología , Factores de Transcripción/genética
13.
Eukaryot Cell ; 7(2): 319-27, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18156290

RESUMEN

The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide (PS) capsule and releases copious amounts of PS into cultures and infected tissues. The capsular PS is a major virulence factor that can elicit protective antibody responses. PS recovered from culture supernatants has historically provided an ample and convenient source of material for structural and immunological studies. Two major assumptions in such studies are that the structural features of the exopolysaccharide material faithfully mirror those of capsular PS and that the isolation methods do not change PS properties. However, a comparison of exopolysaccharide made by two isolation techniques with capsular PS stripped from cells with gamma radiation or dimethyl sulfoxide revealed significant differences in glycosyl composition, mass, size, charge, viscosity, circular-dichroism spectra, and reactivity with monoclonal antibodies. Our results strongly suggest that exopolysaccharides and capsular PS are structurally different. A noteworthy finding was that PS made by cetyltrimethylammonium bromide precipitation had a larger mass and a different conformation than PS isolated by concentration and filtration, suggesting that the method most commonly used to purify glucuronoxylomannan alters the PS. Hence, the method used to isolate PS can significantly influence the structural and antigenic properties of the product. Our findings have important implications for current views of the relationship between capsular PS and exopolysaccharides, for the generation of PS preparations suitable for immunological studies, and for the formulation of PS-based vaccines for the prevention of cryptococcosis.


Asunto(s)
Antígenos Fúngicos/metabolismo , Cápsulas Bacterianas/metabolismo , Cryptococcus neoformans/metabolismo , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Anticuerpos Monoclonales/inmunología , Dicroismo Circular , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/efectos de la radiación , Dimetilsulfóxido/farmacología , Ensayo de Inmunoadsorción Enzimática , Rayos gamma , Espectroscopía de Resonancia Magnética , Polisacáridos Bacterianos/inmunología
14.
mBio ; 10(1)2019 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-30602579

RESUMEN

Living organisms are constantly exposed to DNA damage stress caused by endogenous and exogenous events. Eukaryotic cells have evolutionarily conserved DNA damage checkpoint surveillance systems. We previously reported that a unique transcription factor, Bdr1, whose expression is strongly induced by the protein kinase Rad53 governs DNA damage responses by controlling the expression of DNA repair genes in the basidiomycetous fungus Cryptococcus neoformans However, the regulatory mechanism of the Rad53-dependent DNA damage signal cascade and its function in pathogenicity remain unclear. Here, we demonstrate that Rad53 is required for DNA damage response and is phosphorylated by two phosphatidylinositol 3-kinase (PI3K)-like kinases, Tel1 and Mec1, in response to DNA damage stress. Transcriptome analysis revealed that Rad53 regulates the expression of several DNA repair genes in response to gamma radiation. We found that expression of CHK1, another effector kinase involved in the DNA damage response, is regulated by Rad53 and that CHK1 deletion rendered cells highly susceptible to DNA damage stress. Nevertheless, BDR1 expression is regulated by Rad53, but not Chk1, indicating that DNA damage signal cascades mediated by Rad53 and Chk1 exhibit redundant and distinct functions. We found that perturbation of both RAD53 and CHK1 attenuated the virulence of C. neoformans, perhaps by promoting phagosome maturation within macrophage, reducing melanin production, and increasing susceptibility to oxidative stresses. Furthermore, deletion of both RAD53 and CHK1 increased susceptibility to certain antifungal drugs such as amphotericin B. This report provides insight into the regulatory mechanism of fungal DNA damage repair systems and their functional relationship with fungal virulence and antifungal drug susceptibility.IMPORTANCE Genome instability is detrimental for living things because it induces genetic disorder diseases and transfers incorrect genome information to descendants. Therefore, living organisms have evolutionarily conserved signaling networks to sense and repair DNA damage. However, how the DNA damage response pathway is regulated for maintaining the genome integrity of fungal pathogens and how this contributes to their pathogenicity remain elusive. In this study, we investigated the DNA damage response pathway in the basidiomycete pathogen Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, with an average of 223,100 infections leading to 181,100 deaths reported annually. Here, we found that perturbation of Rad53- and Chk1-dependent DNA damage response pathways attenuated the virulence of C. neoformans and increased its susceptibility to certain antifungal drugs, such as amphotericin B and flucytosine. Therefore, our work paves the way to understanding the important role of human fungal DNA damage networks in pathogenesis and antifungal drug susceptibility.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/crecimiento & desarrollo , Daño del ADN , Reparación del ADN , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Antifúngicos/farmacología , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/efectos de la radiación , Rayos gamma , Perfilación de la Expresión Génica , Fosforilación , Procesamiento Proteico-Postraduccional
15.
Sci Rep ; 9(1): 6385, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31015536

RESUMEN

Cryptococcus neoformans is a basidiomycete fungus that is highly resistant to ionizing radiation and has been identified in highly radioactive environments. Transcription factors (TFs) are master regulators of gene expression by binding to specific DNA sequences within promoters of target genes. A library of 322 signature-tagged gene deletion strains for 155 C. neoformans TF genes has been established. Previous phenome-based functional analysis of the C. neoformans TF mutant library identified key TFs important for various phenotypes, such as growth, differentiation, virulence-factor production, and stress responses. Here, utilizing the established TF mutant library, we identified 5 TFs that are important for radiation sensitivity, including SRE1, BZP2, GAT5, GAT6, and HCM1. Interestingly, BZP2, GAT5 and GAT6 all belong to the GATA-type transcription factors. These factors regulate transcription of nitrogen catabolite repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. In addition to radiation, we found that specific GATA factors are important for other stressors such as rapamycin, fluconazole, and hydroxyurea treatment. Using real-time PCR method, we studied the expression of GATA down-stream genes after radiation exposure and identified that AAP4, AAP5 and URO1 were differentially expressed in the GAT5 and GAT6 mutants compared to the wild type cells. In summary, our data suggest that GATA TFs are important for radiation sensitivity in C. neoformans by regulating specific downstream AAP genes.


Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/efectos de la radiación , Proteínas Fúngicas/genética , Factores de Transcripción GATA/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Tolerancia a Radiación/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Fluconazol/farmacología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Hidroxiurea/farmacología , Mutación/genética , Filogenia , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Sirolimus/farmacología
16.
Antimicrob Agents Chemother ; 52(6): 2232-5, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18378712

RESUMEN

We evaluated the clonogenic survival, membrane permeability, metabolic activity (XTT reduction), and apoptosis (FLICA binding) of Cryptococcus neoformans cells subjected to gamma rays from an external source, and beta and alpha particles delivered to fungal cells by capsule-specific antibody. We found that gamma, beta, and alpha radiation affected cells through different pathways.


Asunto(s)
Partículas alfa , Partículas beta , Permeabilidad de la Membrana Celular/efectos de la radiación , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/efectos de la radiación , Rayos gamma , Animales , Anticuerpos Monoclonales , Apoptosis/efectos de la radiación , Bismuto , Recuento de Colonia Microbiana , Cryptococcus neoformans/crecimiento & desarrollo , Humanos , Radioisótopos , Renio , Sales de Tetrazolio/metabolismo
17.
PLoS Biol ; 3(4): e95, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15760278

RESUMEN

Light inhibits mating and haploid fruiting of the human fungal pathogen Cryptococcus neoformans, but the mechanisms involved were unknown. Two genes controlling light responses were discovered through candidate gene and insertional mutagenesis approaches. Deletion of candidate genes encoding a predicted opsin or phytochrome had no effect on mating, while strains mutated in the white collar 1 homolog gene BWC1 mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with Neurospora crassa WC-1, but lacks the zinc finger DNA binding domain. BWC1 regulates cell fusion and repression of hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other components required for responses to light, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. The C. neoformans Bwc1 and Bwc2 proteins interact in the yeast two-hybrid assay. Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus. These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.


Asunto(s)
Cryptococcus neoformans/crecimiento & desarrollo , Hongos/genética , Luz , Secuencia Conservada , Cruzamientos Genéticos , Cryptococcus neoformans/genética , Cryptococcus neoformans/efectos de la radiación , Hongos/crecimiento & desarrollo , Hongos/efectos de la radiación , Genes Fúngicos , Genoma Fúngico , Datos de Secuencia Molecular , Fotorreceptores Microbianos/genética , Transducción Genética
18.
Clin Cancer Res ; 13(18 Pt 2): 5629s-5635s, 2007 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-17875799

RESUMEN

PURPOSE: The applicability of radioimmunotherapy with organism-specific monoclonal antibodies to treatment of infectious disease in experimental models has been recently shown for fungal, bacterial, and viral infections. To identify the best delivery vehicle for radioimmunotherapy of human pathogenic fungus Cryptococcus neoformans (CN), we have done comparative evaluation of capsular polysaccharide-specific antibodies with IgG1 and IgM isotypes and F(ab')2 and Fab fragments. EXPERIMENTAL DESIGN: 18B7 IgG1 and 13F1 IgM and their isotype-matching controls were radiolabeled with 188Re, and their binding to 24067 and H99 CN strains was evaluated by doing Scatchard and kinetics analyses. The doses delivered during in vitro radioimmunotherapy were estimated using a cellular dosimetry algorithm. The biodistribution of 188Re-labeled 18B7 and 13F1 and of 111In-labeled 18B7 and its F(ab')2 and Fab fragments was done in A/JCr mice systemically infected with 24067 CN strain. RESULTS: 18B7 IgG1 showed superior to 13F1 IgM binding to 24067 CN (Ka=1.7x10(9) mol/L(-1) and 5.4x10(7) mol/L(-1), respectively). Substantial killing of 24067 and H99 CN cells was achieved with 1 microCi 188Re-18B7 (55 cGy dose), whereas no killing was observed for 1 microCi 188Re-13F1 (2 cGy dose). In vivo 188Re-18B7 localized specifically in the lungs of CN-infected mice, whereas uptake of 188Re-13F1 was nonspecific. 111In-F(ab')2 fragments showed higher uptake in the lungs and lower in the liver at the 48-h time point in comparison with intact 111In-18B7. CONCLUSIONS: Comparative evaluation of IgG and IgM and of F(ab')2 and Fab fragments as potential delivery vehicles for radioimmunotherapy of cryptococcal infection strongly suggests that affinity for the target antigen is an important prerequisite for successful targeting of infection in vivo and that in vitro affinity measurements may predict the in vivo efficacy of candidate monoclonal antibodies.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Criptococosis/terapia , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/farmacología , Inmunoglobulina M/farmacología , Inmunotoxinas/farmacología , Radioinmunoterapia , Animales , Anticuerpos Monoclonales/uso terapéutico , Criptococosis/inmunología , Criptococosis/microbiología , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/efectos de la radiación , Sistemas de Liberación de Medicamentos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Inmunoglobulina G/uso terapéutico , Inmunoglobulina M/uso terapéutico , Inmunotoxinas/uso terapéutico , Radioisótopos de Indio/farmacología , Pulmón/citología , Pulmón/metabolismo , Ratones , Polisacáridos/inmunología , Renio/farmacología
19.
Environ Microbiol Rep ; 10(3): 255-263, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29473314

RESUMEN

The aim of this study was to analyse how protracted exposure to X-rays delivered at low dose rates of 0.0032-0.052 kGy h-1 affects the survival and metabolic activity of two microfungi capable of melanogenesis: fast-growing Cryptococcus neoformans (CN) and slow-growing Cryomyces antarcticus (CA). Melanized CN and CA cells survived the protracted exposure better than non-melanized ones, which was consistent with previous reports on the radioprotective role of melanin in these fungi after high dose rate exposures. The survival data were described by the linear quadratic dose response model. The XTT metabolic profiles were practically identical for melanized CN and CA with activity dose-dependent increasing: no changes in the activity of the non-melanized CN and CA were recorded by this assay. In contrast, the MTT assay, which measures the intracellular energy-related processes, recorded an increase in activity of non-melanized CN and CA cells, but not in their melanized counterparts. This could reflect intensive repair processes initiated by the non-melanized cells post exposure. This study suggests that differences in radiation responses between melanized and non-melanized fungal cells occur over a wide range of radiation dose rates.


Asunto(s)
Ascomicetos/efectos de la radiación , Cryptococcus neoformans/efectos de la radiación , Melaninas/biosíntesis , Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/metabolismo , Relación Dosis-Respuesta en la Radiación , Modelos Teóricos , Rayos X
20.
Fungal Biol ; 122(6): 449-456, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29801788

RESUMEN

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.


Asunto(s)
Cryptococcus neoformans/efectos de la radiación , Cryptococcus neoformans/ultraestructura , Melaninas/fisiología , Tolerancia a Radiación , Protectores contra Radiación , Partículas alfa/efectos adversos , Pared Celular/efectos de la radiación , Deuterio/efectos adversos , Rayos gamma/efectos adversos , Microscopía Electrónica de Transmisión , Protección Radiológica
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