Mecanismos moleculares de la Inducción de la esporulación en Aspergillus nidulans

163 p.-47 fig.-5 tab.Las actividades biológicas de los hongos filamentosos han suscitado gran interés en diferentes campos, como la industria alimentaria o en la obtención de metabolitos. El organismo objeto de estudio en esta tesis doctoral, Aspergillus (Emericella) nidulans, es una de las casi 15.000 especies de Ascomicetos, de la clase Eurotiomicetos perteneciente a la familia Trichocomaceae (Thom & Raper, 1945, Pontecorvo et al.,1953). Su genoma de 30,06 millones de pares de bases está organizado en 8 cromosomas y alberga alrededor de 10.700 genes (Galagan et al., 2005). Cabe destacar que A. nidulans constituye un modelo genético de referencia para otros miembros de su género de gran interés económico, como A. oryzae o A. flavus, o en medicina, como A. fumigatus. Su capacidad para formar esporas sexuales mediante meiosis, la existencia de un ciclo sexual y el carácter homotálico (Dyer et al., 2003), o su utilización en estudios genéticos y de biología celular han facilitado su uso como modelo experimental para alcanzar una mejor comprensión de varios procesos biológicos. También ha sido objeto de estudios como la recombinación y reparación de ADN, control del ciclo celular y de metabolismo primario y secundario (Nierman et al., 2005).El ciclo de vida de A. nidulans se caracteriza por la presencia de cuatro estadios diferenciados (Fig I1; (Adams et al., 1998, Todd et al., 2007b, Todd et al., 2007a)): crecimiento vegetativo, reproducción asexual, reproducción sexual y el ciclo parasexual. El ciclo comienza con la germinación de una conidiospora mononucleada o una ascoespora binucleada, la cual da lugar a un tubo de germinación que crece de forma filamentosa como una hifa vegetativa haploide. Dicha germinación se inicia con el establecimiento de un punto de polaridad determinado de la espora/ascoespora (Harris, 1997, Momany, 2002, Harris & Momany, 2004), desplazando hasta ese punto toda la maquinaria requerida para la extensión de la hifa (D'Enfert, 1997, Osherov & May, 2001). Así, mediante crecimiento polar y adición de nuevo material en la punta (Momany, 2002), las hifas se extienden dando lugar a sincitios multinucleados, formando un entramado de hifas interconectadas denominado micelio.Proyecto BFU2004-03499/BMC del Ministerio de Educación y Ciencia ; Proyecto IT393-10 del Gobierno Vasco y Beca PFPI.Peer reviewe

Transcriptional changes in the transition from vegetative cells to asexual development in the model fungus Aspergillus nidulans

32 p.-6 fig.-2 tab.Morphogenesis encompasses programmed changes in gene expression that lead to the development of specialized cell types. In the model fungus Aspergillus nidulans, asexual development involves the formation of characteristic cell types, collectively known as the conidiophore. With the aim of determining the transcriptional changes that occur upon induction of asexual development, we have applied massive mRNA sequencing to compare the expression pattern of 19-h-old submerged vegetative cells (hyphae) with that of similar hyphae after exposure to the air for 5 h. We found that the expression of 2,222 (20.3%) of the predicted 10,943 A. nidulans transcripts was significantly modified after air exposure, 2,035 being downregulated and 187 upregulated. The activation during this transition of genes that belong specifically to the asexual developmental pathway was confirmed. Another remarkable quantitative change occurred in the expression of genes involved in carbon or nitrogen primary metabolism. Genes participating in polar growth or sexual development were transcriptionally repressed, as were those belonging to the HogA/SakA stress response mitogen-activated protein (MAP) kinase pathway. We also identified significant expression changes in several genes purportedly involved in redox balance, transmembrane transport, secondary metabolite production, or transcriptional regulation, mainly binuclear-zinc cluster transcription factors. Genes coding for these four activities were usually grouped in metabolic clusters, which may bring regulatory implications for the induction of asexual development. These results provide a blueprint for further stage-specific gene expression studies during conidiophore development. © 2013, American Society for Microbiology. All Rights Reserved.This work has been supported by the Basque Government through grant (IT393-10) and the Ministerio de Economía y Competitividad (formerly Ministerio de Ciencia e Innovación) through grant (BFU2010-17528) to U.U., grant (BFU2009- 08701) to E.A.E. and grants from the German Science Foundation (DFG Fi 459), the Fonds der Chemischen Industrie, the Baden-Württemberg Stiftung, and the Centre for Functional Nanostructures to R.F. A. G. is now a contract researcher from The University of The Basque Country. J.R. was a postdoctoral fellow of the Ministerio de Ciencia e Innovación. O.E is a contract researcher associated to grant BFU2010- 17528.Peer Reviewe

<i>In vitro</i> antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19

BackgroundCurrent approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.MethodsSARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.ResultsDaclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.ConclusionsDaclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy

Defining the transcriptional responses of Aspergillus nidulans to cation/alkaline pH stress and the role of the transcription factor SltA

18 p.-7 fig.Fungi have developed the ability to overcome extreme growth conditions and thrive in hostile environments. The model fungus Aspergillus nidulans tolerates, for example, ambient alkalinity up to pH 10 or molar concentrations of multiple cations. The ability to grow under alkaline pH or saline stress depends on the effective function of at least three regulatory pathways mediated by the zinc-finger transcription factor PacC, which mediates the ambient pH regulatory pathway, the calcineurin-dependent CrzA and the cation homeostasis responsive factor SltA. Using RNA sequencing, we determined the effect of external pH alkalinization or sodium stress on gene expression. The data show that each condition triggers transcriptional responses with a low degree of overlap. By sequencing the transcriptomes of the null mutant, the role of SltA in the above-mentioned homeostasis mechanisms was also studied. The results show that the transcriptional role of SltA is wider than initially expected and implies, for example, the positive control of the PacC-dependent ambient pH regulatory pathway. Overall, our data strongly suggest that the stress response pathways in fungi include some common but mostly exclusive constituents, and that there is a hierarchical relationship among the main regulators of stress response, with SltA controlling pacC expression, at least in A. nidulans.Work at CIB-CSIC was funded by MINECO (BFU2015-66806-R to E. A.E) and MICIU/AEI (RTI2018-094263-B-100) to E. A. E (both partially supported by FEDER, EU). Work at the UPV/EHU lab was funded by UPV/EHU grants PPGA19/08 and GIU19/014 to O. E and the Basque Government grant Elkartek19/72 (to Professor María Teresa Dueñas).I. P and E. R. held research contracts associated with grants RTI2018-094263-B-100 and BFU2015-66806-R,respectively.We acknowledge the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI) for enabling this open access publication through a Publish and Read deal with the Microbiology Society.Peer reviewe

Elucidation of functional markers from Aspergillus nidulans developmental regulator FlbB and their phylogenetic distribution

Aspergillus nidulans is a filamentous fungus widely used as a model for biotechnological and clinical research. It is also used as a platform for the study of basic eukaryotic developmental processes. Previous studies identified and partially characterized a set of proteins controlling cellular transformations in this ascomycete. Among these proteins, the bZip type transcription factor FlbB is a key regulator of reproduction, stress responses and cell-death. Our aim here was the prediction, through various bioinformatic methods, of key functional residues and motifs within FlbB in order to inform the design of future laboratory experiments and further the understanding of the molecular mechanisms that control fungal development. A dataset of FlbB orthologs and those of its key interaction partner FlbE was assembled from 40 members of the Pezizomycotina. Unique features were identified in each of the three structural domains of FlbB. The N-terminal region encoded a bZip transcription factor domain with a novel histidine-containing DNA binding motif while the dimerization determinants exhibited two distinct profiles that segregated by class. The C-terminal region of FlbB showed high similarity with the AP-1 family of stress response regulators but with variable patterns of conserved cysteines that segregated by class and order. Motif conservation analysis revealed that nine FlbB orthologs belonging to the Eurotiales order contained a motif in the central region that could mediate interaction with FlbE. The key residues and motifs identified here provide a basis for the design of follow-up experimental investigations. Additionally, the presence or absence of these residues and motifs among the FlbB orthologs could help explain the differences in the developmental programs among fungal species as well as define putative complementation groups that could serve to extend known functional characterizations to other speciesThis work was supported by the Basque Government(http://www.hezkuntza.ejgv.euskadi.net) through grant IT393-10 to U.U. and by the Spanish Ministerio de Educacio´n y Ciencia (www.mec.es) through grant BFU2009-08701 to E.A.E. M.S.C. was a contract researcher of the Ikerbasque program of the Basque Government (www.ikerbasque.net). O.E. was a contract researcher of the University of the Basque Country (www.ehu.es). A.G. held a predoctoral fellowship frommthe Basque Government.Peer reviewe

Boron Tolerance in Aspergillus nidulans Is Sustained by the SltA Pathway Through the SLC-Family Transporters SbtA and SbtB

Microbial cells interact with the environment by adapting to external changes. Signal transduction pathways participate in both sensing and responding in the form of modification of gene expression patterns, enabling cell survival. The filamentous fungal-specific SltA pathway regulates tolerance to alkalinity, elevated cation concentrations and, as shown in this work, also stress conditions induced by borates. Growth of sltA− mutants is inhibited by increasing millimolar concentrations of boric acid or borax (sodium tetraborate). In an attempt to identify genes required for boron-stress response, we determined the boric acid or borax-dependent expression of sbtA and sbtB, orthologs of Saccharomyces cerevisiae bor1, and a reduction in their transcript levels in a ΔsltA mutant. Deletion of sbtA, but mainly that of sbtB, decreased the tolerance to boric acid or borax. In contrast, null mutants of genes coding for additional transporters of the Solute Carrier (SLC) family, sB, sbtD or sbtE, showed an unaltered growth pattern under the same stress conditions. Taken together, our results suggest that the SltA pathway induces, through SbtA and SbtB, the export of toxic concentrations of borates, which have largely recognized antimicrobial properties.Work at CIB-CSIC was funded by MINECO project BFU2012-33142 and MINECO/FEDER/EU project BFU2015-66806-R to E.A.E. Work at the UPV/EHU lab was funded by the Basque Government (grant IT599-13 to U.U.) and UPV/EHU (grant EHUA15/08 to O.E.). M.V. held a contract under project BFU2012-33142.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Aspergillus nidulans asexual development: making the most of cellular modules

8 páginas, 4 figuras -- PAGS nros. 569-576Asexual development in Aspergillus nidulans begins in superficial hyphae as the programmed emergence of successive pseudohyphal modules, collectively known as the conidiophore, and is completed by a layer of specialized cells (phialides) giving rise to chains of aerial spores. A discrete number of regulatory factors present in hyphae play different stage-specific roles in pseudohyphal modules, depending on their cellular localization and protein–protein interactions. Their multiple roles include the timely activation of a sporulation-specific pathway that governs phialide and spore formation. Such functional versatility provides for a new outlook on morphogenetic change and the ways we should study itThis work was supported by the Basque Government through grant IT393-10 to U.U. and by the Spanish Ministerio de Educación y Ciencia through grant BFU2009-08701 to E.A.E. O.E. is a contract researcher at The University of The Basque Country. A.G. held a predoctoral fellowship from the Basque GovernmentPeer reviewe

Running title: Evolution of asexual developmental regulators

46 p.-4 fig.-2 tab.-1 fig. supl.Complex multicellularity (CM) is characterized by the generation of three-dimensional structures that follow a genetically controlled program. CM emerged at least five times in evolution, one of them in fungi. There are two types of CM programs in fungi, leading, respectively, to the formation of sexual or asexual spores. Asexual spores foment the spread of mycoses, as they are the main vehicle for dispersion. In spite of this key dependence, there is great morphological diversity of asexual multicellular structures in fungi. To advance the understanding of the mechanisms that control initiation and progression of asexual CM and how they can lead to such a remarkable morphological diversification, we studied 503 fungal proteomes, representing all phyla and subphyla, and most known classes. Conservation analyses of 33 regulators of asexual development suggest stepwise emergence of transcription factors. While velvet proteins constitute one of the most ancient systems, the central regulator BrlA emerged late in evolution (with the class Eurotiomycetes). Some factors, such as MoConX4, seem to be species-specific. These observations suggest that the emergence and evolution of transcriptional regulators rewire transcriptional networks. This process could reach the species level, resulting in a vast diversity of morphologies.Work at the UPV/EHU lab was funded by the University of the Basque Country (grant EHUA15/08). Work at CIB-CSIC was funded by MINECO/FEDER/EU (grant BFU2015-66806-R).Peer reviewe

Boron Tolerance in Aspergillus nidulans Is Sustained by the SltA Pathway Through the SLC-Family Transporters SbtA and SbtB

Microbial cells interact with the environment by adapting to external changes. Signal transduction pathways participate in both sensing and responding in the form of modification of gene expression patterns, enabling cell survival. The filamentous fungal-specific SltA pathway regulates tolerance to alkalinity, elevated cation concentrations and, as shown in this work, also stress conditions induced by borates. Growth of sltA mutants is inhibited by increasing millimolar concentrations of boric acid or borax (sodium tetraborate). In an attempt to identify genes required for boron-stress response, we determined the boric acid or borax-dependent expression of sbtA and sbtB, orthologs of Saccharomyces cerevisiae bor1, and a reduction in their transcript levels in a D sltA mutant. Deletion of sbtA, but mainly that of sbtB, decreased the tolerance to boric acid or borax. In contrast, null mutants of genes coding for additional transporters of the Solute Carrier (SLC) family, sB, sbtD or sbtE, showed an unaltered growth pattern under the same stress conditions. Taken together, our results suggest that the SltA pathway induces, through SbtA and SbtB, the export of toxic concentrations of borates, which have largely recognized antimicrobial properties.Work at CIB-CSIC was funded by MINECO project BFU2012-33142 and MINECO/FEDER/EU project BFU2015-66806-R to E.A.E. Work at the UPV/EHU lab was funded by the Basque Government (grant IT599-13 to U.U.) and UPV/EHU (grant EHUA15/08 to O.E.). M.V. held a contract under project BFU2012-33142. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Gmca is a putative glucose-methanol-choline oxidoreductase required for the induction of asexual development in aspergillus nidulans

Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation. © 2012 Etxebeste et al.This work has been supported by the Basque Government (SA-2010/00105 and IT393-10) and Ministerio de Ciencia e Innovación (BFU2010-17528) to U.U. and to E.A.E. through grant BFU2009-08701. O.E was a contract researcher of The University of The Basque Country and is now associated to grant BFU2010- 17528. E.H.-G. and A.G. held a predoctoral fellowship from the Basque Government, and M.S.C. was a contract researcher of the Ikerbasque program of the Basque Government (www.ikerbasque.net).Peer Reviewe