Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Más filtros

Métodos Terapéuticos y Terapias MTCI
Bases de datos
Tipo del documento
Intervalo de año de publicación
1.
mBio ; 11(3)2020 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-32398317

RESUMEN

Aspergillus fumigatus can cause a variety of lung diseases in immunocompromised patients, including life-threatening invasive aspergillosis. There are only three main classes of antifungal drugs currently used to treat aspergillosis, and antifungal resistance is increasing. Experimental results in fungal biology research are usually obtained as average measurements across whole populations while ignoring what is happening at the single cell level. In this study, we show that conidia with the same genetic background in the same cell population at a similar developmental stage show heterogeneity in their cell wall labeling at the single cell level. We present a rigorous statistical method, newly applied to quantify the level of cell heterogeneity, which allows for direct comparison of the heterogeneity observed between treatments. We show the extent of cell wall labeling heterogeneity in dormant conidia and how the level of heterogeneity changes during germination. The degree of heterogeneity is influenced by deletions of cell wall synthesizing genes and environmental conditions, including medium composition, method of inoculation, age of conidia, and the presence of antifungals. This heterogeneity results in subpopulations of germinating conidia with heterogeneous fitness to the antifungal caspofungin, which targets cell wall synthesis and heterogeneous sensitivity of dormant conidia to phagocytosis by macrophages.IMPORTANCE The fungus Aspergillus fumigatus can cause invasive lung diseases in immunocompromised patients resulting in high mortality. Treatment using antifungal compounds is often unsuccessful. Average population measurements hide what is happening at the individual cell level. We set out to test what impact individual differences between the cell walls of fungal conidia have on their behavior. We show that a population of cells having the same genetic background gives rise to subpopulations of cells that exhibit distinct behavior (phenotypic heterogeneity). This cell heterogeneity is dependent on the strain type, gene deletions, cell age, and environmental conditions. By looking at the individual cell level, we discovered subpopulations of cells that show differential fitness during antifungal treatment and uptake by immune cells.


Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Pared Celular/química , Fagocitosis/efectos de los fármacos , Animales , Farmacorresistencia Fúngica , Regulación Fúngica de la Expresión Génica , Ratones , Células RAW 264.7 , Análisis de la Célula Individual , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética
2.
Artículo en Inglés | MEDLINE | ID: mdl-30397071

RESUMEN

Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.


Asunto(s)
Antifúngicos/uso terapéutico , Aspergillus fumigatus/efectos de los fármacos , Brefeldino A/uso terapéutico , Fusión Celular/métodos , Farmacorresistencia Fúngica Múltiple/genética , Itraconazol/uso terapéutico , Factores de Ribosilacion-ADP/genética , Aspergilosis/tratamiento farmacológico , Aspergillus fumigatus/genética , Aspergillus fumigatus/fisiología , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Técnicas de Inactivación de Genes , Humanos , Pruebas de Sensibilidad Microbiana , ATPasas de Translocación de Protón Mitocondriales/genética
3.
Invest Ophthalmol Vis Sci ; 57(14): 6367-6373, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27898982

RESUMEN

Purpose: Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. Methods: Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. Results: The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. Conclusions: A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.


Asunto(s)
Córnea/patología , Reactivos de Enlaces Cruzados/uso terapéutico , Infecciones Fúngicas del Ojo/tratamiento farmacológico , Fusariosis/tratamiento farmacológico , Queratitis/tratamiento farmacológico , Microscopía Confocal/métodos , Recolección de Tejidos y Órganos/métodos , Anciano , Anciano de 80 o más Años , Cadáver , Córnea/efectos de los fármacos , Infecciones Fúngicas del Ojo/diagnóstico , Infecciones Fúngicas del Ojo/microbiología , Femenino , Fusariosis/diagnóstico , Fusariosis/microbiología , Fusarium/aislamiento & purificación , Humanos , Queratitis/diagnóstico , Queratitis/microbiología , Masculino , Persona de Mediana Edad , Fototerapia/métodos , Donantes de Tejidos , Resultado del Tratamiento
4.
Antimicrob Agents Chemother ; 59(8): 4946-55, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26055379

RESUMEN

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.


Asunto(s)
Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Equinocandinas/farmacología , Fosforilación/fisiología , Anidulafungina , Antifúngicos/farmacología , Caspofungina , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Proteínas Fúngicas/metabolismo , Lipopéptidos/farmacología , Micafungina
5.
Eukaryot Cell ; 9(9): 1374-82, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20622001

RESUMEN

The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca(2+) signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca(2+) concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca(2+) ([Ca(2+)](c)). The Ca(2+) signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid], a Ca(2+) selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca(2+) homeostasis. These results indicate that extracellular Ca(2+) was the major source of these PAF-induced effects. The L-type Ca(2+) channel blocker diltiazem disrupted Ca(2+) homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca(2+) signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca(2+)](c) resting level. However, experiments with an aequorin-expressing Deltacch-1 deletion strain of N. crassa indicated that the L-type Ca(2+) channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca(2+)](c) resting level. This study is the first demonstration of the perturbation of fungal Ca(2+) homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.


Asunto(s)
Antifúngicos/farmacología , Calcio/metabolismo , Proteínas Fúngicas/farmacología , Neurospora crassa/efectos de los fármacos , Neurospora crassa/metabolismo , Antifúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/genética , Neurospora crassa/crecimiento & desarrollo , Penicillium chrysogenum/química , Penicillium chrysogenum/metabolismo , Transducción de Señal/efectos de los fármacos
6.
Mol Cell ; 13(3): 427-34, 2004 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-14967149

RESUMEN

Methylation of cytosines silences transposable elements and selected cellular genes in mammals, plants, and some fungi. Recent findings have revealed mechanistic connections between DNA methylation and features of specialized condensed chromatin, "heterochromatin." In Neurospora crassa, DNA methylation depends on trimethylation of Lys9 in histone H3 by DIM-5. Heterochromatin protein HP1 binds methylated Lys9 in vitro. We therefore investigated the possibility that a Neurospora HP1 homolog reads the methyl-Lys9 mark to signal DNA methylation. We identified an HP1 homolog and showed that it is essential for DNA methylation, is localized to heterochromatic foci, and that this localization is dependent on the catalytic activity of DIM-5. We conclude that HP1 serves as an adaptor between methylated H3 Lys9 and the DNA methylation machinery. Unlike mutants that lack DNA methyltransferase, mutants with defects in the HP1 gene hpo exhibit severe growth defects, suggesting that HP1 is required for processes besides DNA methylation.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Proteínas Fúngicas/metabolismo , N-Metiltransferasa de Histona-Lisina , Neurospora crassa/enzimología , Dominio Catalítico/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/aislamiento & purificación , ADN/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Heterocromatina/genética , Histona Metiltransferasas , Lisina/metabolismo , Metiltransferasas/genética , Datos de Secuencia Molecular , Neurospora crassa/genética , Proteína Metiltransferasas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA