Nutrient acquisition and metabolic adaptation in the context of Candida albicans virulence

Candida albicans is an opportunistic human fungal pathogen. Normally a harmless commensal and part of the microbiota of healthy humans, it can cause superficial to life-threatening systemic infections under distinct circumstances. Central for this switch is a variety of fungal pathogenicity mechanisms. Amongst them is a remarkable metabolic plasticity, which is intricately linked with other central fungal virulence traits like the yeast-to-hyphae switch. The chief aim of this thesis was to investigate three different aspects of these connections. The first part focused on nutrient acquisition on the example of proline – not only an especially valuable nutrient source for C. albicans but also a potent morphogenetic stimulus. In this study Gnp2 was identified as a specialized proline permease. Further analysis revealed an essential role for this permease for proline-induced morphogenesis and fungal resistance against macrophage killing and exposure to reactive oxygen species. In the second part a combinatorial approach of transcriptional and metabolic profiling was utilized to examine the metabolic adaptation of C. albicans to varying degrees of amino acid availability. Thereby, a repressive activity of the central amino acid metabolism regulator Stp2 was found on a metabolic gene cluster, which is required for the assimilation of hydroxybenzenes. Together with subsequent phenotypical analyses these findings suggested a so far unknown link between the metabolism of aromatic amino acids and hydroxybenzenes in C. albicans. Lastly, metabolic changes associated with the fungal switch from yeast to hyphal growth were investigated. A variety of morphotype-specific activities of metabolic pathways was identified, most notably including a stimulus-independent activation of the de novo sphingolipid biosynthesis in C. albicans hyphae. Further, by the pharmacological inhibition of this pathway its essential role for proper filamentation was verified

Yarrowia lipolytica : an industrial workhorse

Yarrowia lipolytica is one of the most extensively studied ‘‘non-conventional’’ yeasts, being a strictly aerobic microorganism capable of producing important metabolites and having an intense secretory activity, which justifies efforts to use it in industry (as a biocatalyst), in molecular biology and in genetics studies. Moreover, Y. lipolytica has been considered an adequate model for dimorphism studies in yeasts. Yarrowia lipolytica presents the ability to grow on Olive Mill Wastewater (OMW) as well as to degradate organic compounds, including aliphatic and aromatic hydrocarbons, often accompanied by biosurfactants production. One of the most important products secreted by this microorganism is lipase which can be exploited for several applications in the detergent, food, pharmaceutical, and environmental industries. In addition, Y. lipolytica is able to produce citric acid and aroma from a variety of carbon sources, including sugars, alkanes, plant oils, starch hydrolysates, ethanol, and glycerol. Thus, this chapter presents an overview of Yarrowia lipolytica features and its major biotechnological applications

Modifying Yeast Tolerance to Inhibitory Conditions of Ethanol Production Processes

Saccharomyces cerevisiae strains having a broad range of substrate utilization, rapid substrate consumption, and conversion to ethanol, as well as good tolerance to inhibitory conditions are ideal for cost-competitive ethanol production from lignocellulose. A major drawback to directly design S. cerevisiae tolerance to inhibitory conditions of lignocellulosic ethanol production processes is the lack of knowledge about basic aspects of its cellular signaling network in response to stress. Here, we highlight the inhibitory conditions found in ethanol production processes, the targeted cellular functions, the key contributions of integrated -omics analysis to reveal cellular stress responses according to these inhibitors, and current status on design-based engineering of tolerant and efficient S. cerevisiae strains for ethanol production from lignocellulose

Adaptive laboratory evolution principles and applications for biotechnology

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.(VLID)90682

The Role of Compartmentalized Metabolism in Cellular Metal Homeostasis

The building blocks of cells are usually thought to be DNA, RNA, and proteins. However, life, as we know it, is not possible without iron. While the list of iron’s vital cellular functions is extensive, iron is also quite cytotoxic. Thus, great pains have been taken, at the gene regulation level, to assure that a cell has sufficient iron to copy its genome and power its mitochondria but not too much to damage its membranes with lipid peroxides. Cellular organelles, which accompanied the rise of atmospheric oxygen and an increased need for iron, also play a key role in iron homeostasis. Mitochondria are the sites of iron assimilation whereas lysosomes, with their v-ATPase-generated acidic lumens, are responsible for iron uptake and rely. In the first part of this work, I focused on lysosomes. Unbiased genetic screens performed on cells grown at sub-lethal levels of lysosomal pH inhibition identified several important metabolic pathways in this context. These included central carbon metabolism, cholesterol synthesis and iron homeostasis. While, cells starve for cholesterol and iron, only iron supplementation was necessary and sufficient to restore cell proliferation upon genetic or pharmacologic v-ATPase inhibition. Interestingly, iron supplementation rescued cell viability independent of lysosomal associated functions including signaling and endocytosis. It did, however, reverse changes resulting from low cellular iron including destabilized iron sulfur cluster proteins, induced hypoxia signaling, and impaired respiration. Finally, due to compromised aconitase activity, I identified an increased dependence on pyruvate-derived citrated as a metabolic ramification of lysosomal dysfunction. Taken together, this strongly argued that providing cellular iron is the essential function of lysosomal acidity for cell proliferation In the second part of this work, I explored other organelles in the setting of altered cellular iron. Using unbiased genetic screens, I found that iron chelation necessitates a fully intact mitochondrial iron import system. In this context, Golgi manganese uptake and storage were also essential. Because chemical or genetic induced manganese overload phenocopied iron starvation, cells were more sensitives to iron starvation and resistant to iron overload and ferroptosis. As mitochondria are the main sites of iron assimilation, I also characterized mitochondrial proteomic changes upon altered cellular iron. Here, I identified a mitochondrial solute transporter, SLC25A39, whose protein stability was proportional to cellular iron levels. Further investigation found a key role for this protein in maintaining mitochondrial GSH levels. Finally, I found that damaged or liberated iron sulfur clusters, rather than free iron, determine SLC25A39 stability. This finding may also represent an organelleautonomous regulatory loop in which mitochondria coordinate GSH and iron homeostasis

Transcription factor mediated control of anthocyanin biosynthesis in vegetative tissues

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology

Copper Homeostasis in the Filamentous Ascomycete Aspergillus nidulans.

237 p.El objetivo de esta tesis doctoral ha sido el estudio de la homeostasis del cobre en el hongo filamentoso Aspergillus nidulans. El cobre es un oligoelemento necesario para la vida, pero en cantidades superiores a las necesarias tiene efectos nocivos. Debido a esta propiedad el cobre ha sido utilizado como antimicrobiano en la agricultura en cantidades inconmensurables generando un problema medioambiental. Los resultados de la tesis están divididos en tres capítulos: En el primero, por una parte se detallan los resultados de un experimento de RNA-seq que compara el transcriptoma del hongo en condiciones vegetativas y condiciones de toxicidad por cobre, y por la otra, se describe el sistema de internalización de cobre de alta afinidad que posee el organismo, las proteínas responsables de dicha internalización, su regulación y su sublocalización celular. El segundo capítulo se centra en el mecanismo de detoxificación que el hongo posee para evitar un exceso de cobre dentro de la célula. Se describe el método de detoxificación que posee el hongo, la proteína que lleva a cabo el proceso, su regulación, su localización subcelular y la regulación de todo el sistema mediante un factor de transcripción. En el último capítulo, se describe la metodología seguida para buscar posibles inhibidores de posibles ¿targets¿ del sistema de homeostasis del cobre para potenciar el efecto de fungicidas basados en cobre y así poder bajar las dosis de cobre

Saccharomyces cerevisiae nutrient signaling pathways show an unexpected early activation pattern during winemaking

[Background] accharomyces cerevisiae wine strains can develop stuck or sluggish fermentations when nutrients are scarce or suboptimal. Nutrient sensing and signaling pathways, such as PKA, TORC1 and Snf1, work coordinately to adapt growth and metabolism to the amount and balance of the different nutrients in the medium. This has been exhaustively studied in laboratory strains of S. cerevisiae and laboratory media, but much less under industrial conditions.[Results] Inhibitors of such pathways, like rapamycin or 2-deoxyglucose, failed to discriminate between commercial wine yeast strains with different nutritional requirements, but evidenced genetic variability among industrial isolates, and between laboratory and commercial strains. Most signaling pathways involve events of protein phosphorylation that can be followed as markers of their activity. The main pathway to promote growth in the presence of nitrogen, the TORC1 pathway, measured by the phosphorylation of Rps6 and Par32, proved active at the very start of fermentation, mainly on day 1, and ceased soon afterward, even before cellular growth stopped. Transcription factor Gln3, which activates genes subject to nitrogen catabolite repression, was also active for the first hours, even when ammonium and amino acids were still present in media. Snf1 kinase was activated only when glucose was exhausted under laboratory conditions, but was active from early fermentation stages. The same results were generally obtained when nitrogen was limiting, which indicates a unique pathway activation pattern in winemaking. As PKA remained active throughout fermentation, it could be the central pathway that controls others, provided sugars are present.[Conclusions] Wine fermentation is a distinct environmental situation from growth in laboratory media in molecular terms. The mechanisms involved in glucose and nitrogen repression respond differently under winemaking conditions.This work was funded by a grant from the Spanish Ministry of Economy and Competiveness (MINECO; AGL2017-83254-R) to EM and AA. BV was supported by an F.P.U. fellowship from the Spanish Ministry of Education.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

28th Fungal Genetics Conference

Full abstracts from the 28th Fungal Genetics Conference Asilomar, March 17-22, 2015