RESUMEN
The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8-Br-cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen-activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non-functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteína Quinasa C/genética , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Sustitución de Aminoácidos , Antifúngicos/farmacología , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/enzimología , Cafeína/farmacología , Toxina del Cólera/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Proteínas Fúngicas/efectos de los fármacos , Cinética , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Activación Plaquetaria/farmacología , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteína de Unión al GTP rhoA/efectos de los fármacos , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the beta-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.
Asunto(s)
Antifúngicos/farmacología , Apoptosis , Aspergillus nidulans/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Proteínas Fúngicas/farmacología , Antifúngicos/toxicidad , Aspergillus nidulans/ultraestructura , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular , Proteínas Fúngicas/toxicidad , Proteínas de Unión al GTP/metabolismo , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Penicillium , Penicillium chrysogenum/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
The Penicillium chrysogenumantifungal protein PAF is secreted into the supernatant after elimination of a preprosequence. PAF is actively internalized into the hyphae of sensitive molds and provokes growth retardation as well as changes in morphology. Thus far, no information is available on the exact mode of action of PAF, nor on the function of its prosequence in protein activity. Therefore, we sought to investigate the effects of secreted PAF as well as of intracellularly retained pro-PAF and mature PAF on the sensitive ascomycete Aspergillus nidulans, and transformed this model organism by expression vectors containing 5'-sequentially truncated paf-coding sequences under the control of the inducible P. chrysogenum-derived xylanase promoter. Indirect immunofluorescence staining revealed the localization of recombinant PAF predominantly in the hyphal tips of the transformant Xylpaf1 which expressed prepro-PAF, whereas the protein was found to be distributed intracellularly within all segments of hyphae of the transformants Xylpaf2 and Xylpaf3 which expressed pro-PAF and mature PAF, respectively. Growth retardation of Xylpaf1 and Xylpaf3 hyphae was detected by proliferation assays and by light microscopy analysis. Using transmission electron microscopy of ultrathin hyphal sections a marked alteration of the mitochondrial ultrastructure in Xylpaf1 was observed and an elevated amount of carbonylated proteins pointed to severe oxidative stress in this strain. The effects induced by secreted recombinant PAF resembled those evoked by native PAF. The results give evidence that properly folded PAF is a prerequisite for its activity.
Asunto(s)
Antifúngicos/química , Antifúngicos/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Pliegue de Proteína , Aspergillus nidulans/química , Aspergillus nidulans/citología , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus nidulans/ultraestructura , Clonación Molecular , Proteínas Fúngicas/genética , Hifa/química , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mitocondrias/ultraestructura , Penicillium , Proteínas Recombinantes/farmacología , Eliminación de SecuenciaRESUMEN
The Penicillium chrysogenum antifungal protein PAF inhibits the growth of various filamentous fungi. In this study, PAF was found to localize to the cytoplasm of sensitive aspergilli by indirect immunofluorescence staining. The internalization process required active metabolism and ATP and was prevented by latrunculin B, suggesting an endocytotic mechanism.
Asunto(s)
Antifúngicos/metabolismo , Aspergillus/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/metabolismo , Adenosina Trifosfato/metabolismo , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Aspergillus/efectos de los fármacos , Aspergillus/ultraestructura , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/farmacología , Penicillium , Tiazoles/farmacología , TiazolidinasRESUMEN
The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF ( Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.