Showing papers by "Maria João Sousa published in 2021"

Regulation of cell death induced by acetic acid in yeasts

Abstract: Acetic acid has long been considered a molecule of great interest in the yeast research field. It is mostly recognized as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, as well as of lignocellulosic biomass pretreatment. High acetic acid levels are commonly associated with arrested fermentations or with utilization as vinegar in the food industry. Due to its obvious interest to industrial processes, research on the mechanisms underlying the impact of acetic acid in yeast cells has been increasing. In the past twenty years, a plethora of studies have addressed the intricate cascade of molecular events involved in cell death induced by acetic acid, which is now considered a model in the yeast regulated cell death field. As such, understanding how acetic acid modulates cellular functions brought about important knowledge on modulable targets not only in biotechnology but also in biomedicine. Here, we performed a comprehensive literature review to compile information from published studies performed with lethal concentrations of acetic acid, which shed light on regulated cell death mechanisms. We present an historical retrospective of research on this topic, first providing an overview of the cell death process induced by acetic acid, including functional and structural alterations, followed by an in-depth description of its pharmacological and genetic regulation. As the mechanistic understanding of regulated cell death is crucial both to design improved biomedical strategies and to develop more robust and resilient yeast strains for industrial applications, acetic acid-induced cell death remains a fruitful and open field of study.

Biotechnological Importance of Torulaspora delbrueckii: From the Obscurity to the Spotlight.

Abstract: Torulaspora delbrueckii has attracted interest in recent years, especially due to its biotechnological potential, arising from its flavor- and aroma-enhancing properties when used in wine, beer or bread dough fermentation, as well as from its remarkable resistance to osmotic and freezing stresses. In the present review, genomic, biochemical, and phenotypic features of T. delbrueckii are described, comparing them with other species, particularly with the biotechnologically well-established yeast, Saccharomyces cerevisiae. We conclude about the aspects that make this yeast a promising biotechnological model to be exploited in a wide range of industries, particularly in wine and bakery. A phylogenetic analysis was also performed, using the core proteome of T. delbrueckii, to compare the number of homologous proteins relative to the most closely related species, understanding the phylogenetic placement of this species with robust support. Lastly, the genetic tools available for T. delbrueckii improvement are discussed, focusing on adaptive laboratorial evolution and its potential.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

Abstract: The capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the Hgt-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six Hxt-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.

Improvement of Torulaspora delbrueckii genome annotation: towards the exploitation of genomic features of a biotechnologically relevant yeast

Abstract: Saccharomyces cerevisiae is the most commonly used yeast in wine, beer, and bread fermentations. However, Torulaspora delbrueckii has attracted interest in recent years due to its properties, ranging from its ability to produce flavor- and aroma-enhanced wine to its ability to survive longer in frozen dough. In this work, publicly available genomes of T. delbrueckii were explored and their annotation was improved. A total of 32 proteins were additionally annotated for the first time in the type strain CBS1146, in comparison with the previous annotation available. In addition, the annotation of the remaining three T. delbrueckii strains was performed for the first time. eggNOG-mapper was used to perform the functional annotation of the deduced T. delbrueckii coding genes, offering insights into its biological significance, and revealing 24 clusters of orthologous groups (COGs), which were gathered in three main functional categories: information storage and processing (28% of the proteins), cellular processing and signaling (27%), and metabolism (23%). Small intraspecies variability was found when considering the functional annotation of the four available T. delbrueckii genomes. A comparative study was also conducted between the T. delbrueckii genome and those from 386 fungal species, revealing a high number of homologous genes with species from the Zygotorulaspora and Zygosaccharomyces genera, but also with Lachancea and S. cerevisiae. Lastly, the phylogenetic placement of T. delbrueckii was clarified using the core homologs that were found across 204 common protein sequences of 386 fungal species and strains.

The Plasma Membrane at the Cornerstone Between Flexibility and Adaptability: Implications for Saccharomyces cerevisiae as a Cell Factory.

Abstract: In the last decade, microbial-based biotechnological processes are paving the way toward sustainability as they implemented the use of renewable feedstocks. Nonetheless, the viability and competitiveness of these processes are often limited due to harsh conditions such as: the presence of feedstock-derived inhibitors including weak acids, non-uniform nature of the substrates, osmotic pressure, high temperature, extreme pH. These factors are detrimental for microbial cell factories as a whole, but more specifically the impact on the cell's membrane is often overlooked. The plasma membrane is a complex system involved in major biological processes, including establishing and maintaining transmembrane gradients, controlling uptake and secretion, intercellular and intracellular communication, cell to cell recognition and cell's physical protection. Therefore, when designing strategies for the development of versatile, robust and efficient cell factories ready to tackle the harshness of industrial processes while delivering high values of yield, titer and productivity, the plasma membrane has to be considered. Plasma membrane composition comprises diverse macromolecules and it is not constant, as cells adapt it according to the surrounding environment. Remarkably, membrane-specific traits are emerging properties of the system and therefore it is not trivial to predict which membrane composition is advantageous under certain conditions. This review includes an overview of membrane engineering strategies applied to Saccharomyces cerevisiae to enhance its fitness under industrially relevant conditions as well as strategies to increase microbial production of the metabolites of interest.

N-(5-Amino-9H-benzo[a]phenoxazin-9-ylidene)propan-1-aminium chlorides as antifungal agents and NIR fluorescent probes

Abstract: The search for benzo[a]phenoxazines, Nile Blue derivatives, with high antifungal activity and cell labelling capacity based on our previously published work on this type of compounds led us to the design of compounds with specific substituents in the polycyclic system. Thus, in the present work, four new benzo[a]phenoxazinium chlorides, possessing at the 5-position amino or (3-aminopropyl) amino groups and at the 9-position propylamino or dipropylamino groups, were synthesized. Another analogue, with a (3-aminopropyl) amino group at the 5-position, an ethyl amino group at the 9-position and a methyl group at the 10-position of the polycyclic system, was also synthesized for comparison in the studies performed. The fundamental photophysics (absorption and fluorescence emission) was studied in absolute ethanol, water, and other aqueous solutions of different pH values, relevant for the potential biological applications of these compounds. The antiproliferative activity of the synthesized benzo[a]phenoxazinium chlorides was determined using Saccharomyces cerevisiae PYCC 4072 and the microdilution method described for antifungal susceptibility tests in yeast. All compounds revealed antifungal activity, the most active being the one possessing an amino group at the 5-position and an aminopropyl group at the 9-position. The potential as fluorescent probes was evaluated by fluorescence microscopy, using S. cerevisae as a model system of eukaryotic cells, and it was found that the benzo[a]phenoxazinium chlorides stained the cells with preferential accumulation that seems to appear at the vacuolar membrane and/or the perinuclear membrane of the endoplasmatic reticulum.

Novel Nile Blue Analogue Stains Yeast Vacuolar Membrane, Endoplasmic Reticulum, and Lipid Droplets, Inducing Cell Death through Vacuole Membrane Permeabilization.

Abstract: Phenoxazine derivatives such as Nile Blue analogues are assumed to be increasingly relevant in cell biology due to their fluorescence staining capabilities and antifungal and anticancer activities. However, the mechanisms underlying their effects remain poorly elucidated. Using S. cerevisiae as a eukaryotic model, we found that BaP1, a novel 5- and 9-N-substituted benzo[a]phenoxazine synthesized in our laboratory, when used in low concentrations, accumulates and stains the vacuolar membrane and the endoplasmic reticulum. In contrast, at higher concentrations, BaP1 stains lipid droplets and induces a regulated cell death process mediated by vacuolar membrane permeabilization. BaP1 also induced mitochondrial fragmentation and depolarization but did not lead to ROS accumulation, changes in intracellular Ca2+, or loss of plasma membrane integrity. Additionally, our results show that the cell death process is dependent on the vacuolar protease Pep4p and that the vacuole permeabilization results in its translocation from the vacuole to the cytosol. In addition, although nucleic acids are commonly described as targets of benzo[a]phenoxazines, we did not find any alterations at the DNA level. Our observations highlight BaP1 as a promising molecule for pharmacological application, using vacuole membrane permeabilization as a targeted approach.

In vitro neuronal networks show bidirectional axonal conduction with antidromic action potentials effectively depolarizing the soma

Abstract: Recent technological advances are revealing the complex physiology of the axon and challenging long-standing assumptions. Namely, while most action potential (AP) initiation occurs at the axon initial segment in central nervous system neurons, initiation in distal parts of the axon has been shown to occur in both physiological and pathological conditions. However, such ectopic action potential (EAP) activity has not been reported yet in studies using in vitro neuronal networks and its functional role, if exists, is still not clear. Here, we report the spontaneous occurrence of EAPs and effective antidromic conduction in hippocampal neuronal cultures. We also observe a significant fraction of bidirection axonal conduction in dorsal root ganglia neuronal cultures. We set out to investigate and characterize this antidromic propagation via a combination of microelectrode arrays, microfluidics, advanced data analysis and in silico studies. We show that EAPs and antidromic conduction can occur spontaneously, and also after distal axotomy or physiological changes in the axon biochemical environment. Importantly, EAPs may carry information (as orthodromic action potentials do) and can have a functional impact on the neuron, as they consistently depolarize the soma. Plasticity or gene transduction mechanisms triggered by soma depolarization can, therefore, be also affected by these antidromic action potentials/EAPs. Finally, we show that this bidirectional axonal conduction is asymmetrical, with antidromic conduction being slower than orthodromic. Via computational modeling, we show that the experimental difference can be explained by axonal morphology. Altogether, these findings have important implications for the study of neuronal function in vitro, reshaping completely our understanding on how information flows in neuronal cultures.