Sandra Costa dos Santos

YEASTRACT: providing a programmatic access to curated transcriptional regulatory associations in Saccharomyces cerevisiae through a web services interface

The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT) information system (http://www.yeastract.com) was developed to support the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in June 2010, this database contains over 48 200 regulatory associations between transcription factors (TFs) and target genes, including 298 specific DNA-binding sites for 110 characterized TFs. All regulatory associations stored in the database were revisited and detailed information on the experimental evidences that sustain those associations was added and classified as direct or indirect evidences. The inclusion of this new data, gathered in response to the requests of YEASTRACT users, allows the user to restrict its queries to subsets of the data based on the existence or not of experimental evidences for the direct action of the TFs in the promoter region of their target genes. Another new feature of this release is the availability of all data through a machine readable web-service interface. Users are no longer restricted to the set of available queries made available through the existing web interface, and can use the web service interface to query, retrieve and exploit the YEASTRACT data using their own implementation of additional functionalities. The YEASTRACT information system is further complemented with several computational tools that facilitate the use of the curated data when answering a number of important biological questions. Since its first release in 2006, YEASTRACT has been extensively used by hundreds of researchers from all over the world. We expect that by making the new data and services available, the system will continue to be instrumental for yeast biologists and systems biology researchers.

The YEASTRACT database: an upgraded information system for the analysis of gene and genomic transcription regulation in Saccharomyces cerevisiae

The YEASTRACT (http://www.yeastract.com) information system is a tool for the analysis and prediction of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in June 2013, this database contains over 200 000 regulatory associations between transcription factors (TFs) and target genes, including 326 DNA binding sites for 113 TFs. All regulatory associations stored in YEASTRACT were revisited and new information was added on the experimental conditions in which those associations take place and on whether the TF is acting on its target genes as activator or repressor. Based on this information, new queries were developed allowing the selection of specific environmental conditions, experimental evidence or positive/negative regulatory effect. This release further offers tools to rank the TFs controlling a gene or genome-wide response by their relative importance, based on (i) the percentage of target genes in the data set; (ii) the enrichment of the TF regulon in the data set when compared with the genome; or (iii) the score computed using the TFRank system, which selects and prioritizes the relevant TFs by walking through the yeast regulatory network. We expect that with the new data and services made available, the system will continue to be instrumental for yeast biologists and systems biology researchers.

Drug:H+ antiporters in chemical stress response in yeast

The emergence of widespread multidrug resistance (MDR) is a serious challenge for therapeutics, food-preservation and crop protection. Frequently, MDR is a result of the action of drug-efflux pumps, which are able to catalyze the extrusion of unrelated chemical compounds. This review summarizes the current knowledge on the Saccharomyces cerevisiae drug:H+ antiporters of the major facilitator superfamily (MFS), a group of MDR transporters that is still characterized poorly in eukaryotes. Particular focus is given here to the physiological role and expression regulation of these transporters, while we provide a unified view of new data emerging from functional genomics approaches. Although traditionally described as drug pumps, evidence reviewed here corroborates the hypothesis that several MFS-MDR transporters might have a natural substrate and that drug transport might occur only fortuitously or opportunistically. Their role in MDR might even result from the transport of endogenous metabolites that affect the partition of cytotoxic compounds indirectly. Finally, the extrapolation of the gathered knowledge on the MDR phenomenon in yeast to pathogenic fungi and higher eukaryotes is discussed.

An amphibian‐derived, cationic, α‐helical antimicrobial peptide kills yeast by caspase‐independent but AIF‐dependent programmed cell death

The dermaseptins are a family of antimicrobial peptides from the tree‐frog Phyllomedusa sauvagii. Yeast exposed to dermaseptin S3(1‐16), a truncated derivative of dermaseptin S3 with full activity, showed diagnostic markers of yeast apoptosis: the appearance of reactive oxygen species and fragmentation of nuclear DNA. This process was independent of the yeast caspase, Yca1p. Screening of a non‐essential gene deletion collection in yeast identified genes that conferred resistance to dermaseptin S3(1‐16): izh2Δ, izh3Δ, stm1Δ and aif1Δ, all known to be involved in regulating yeast apoptosis. The appearance of apoptotic markers was reduced in these strains when exposed to the peptide. Dermaseptin S3(1‐16) was shown to interact with DNA, and cause DNA damage in vivo, a process known to trigger apoptosis. Supporting this, a dermaseptin S3(1‐16) affinity column specifically purified Stm1p, Mre11p and Htb2p; DNA‐binding proteins implicated in yeast apoptosis and DNA repair. Thus, amphibians may have evolved a mechanism to induce cell suicide in invading fungal pathogens.

Yeast Toxicogenomics: Genome-Wide Responses to Chemical Stresses with Impact in Environmental Health, Pharmacology, and Biotechnology

The emerging transdisciplinary field of Toxicogenomics aims to study the cell response to a given toxicant at the genome, transcriptome, proteome, and metabolome levels. This approach is expected to provide earlier and more sensitive biomarkers of toxicological responses and help in the delineation of regulatory risk assessment. The use of model organisms to gather such genomic information, through the exploitation of Omics and Bioinformatics approaches and tools, together with more focused molecular and cellular biology studies are rapidly increasing our understanding and providing an integrative view on how cells interact with their environment. The use of the model eukaryote Saccharomyces cerevisiae in the field of Toxicogenomics is discussed in this review. Despite the limitations intrinsic to the use of such a simple single cell experimental model, S. cerevisiae appears to be very useful as a first screening tool, limiting the use of animal models. Moreover, it is also one of the most interesting systems to obtain a truly global understanding of the toxicological response and resistance mechanisms, being in the frontline of systems biology research and developments. The impact of the knowledge gathered in the yeast model, through the use of Toxicogenomics approaches, is highlighted here by its use in prediction of toxicological outcomes of exposure to pesticides and pharmaceutical drugs, but also by its impact in biotechnology, namely in the development of more robust crops and in the improvement of yeast strains as cell factories.

Long-term colonization of the cystic fibrosis lung by Burkholderia cepacia complex bacteria: epidemiology, clonal variation, and genome-wide expression alterations

Long-term respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients generally lead to a more rapid decline in lung function and, in some cases, to a fatal necrotizing pneumonia known as the “cepacia syndrome.” Bcc bacteria are ubiquitous in the environment and are recognized as serious opportunistic pathogens that are virtually impossible to eradicate from the CF lung, posing a serious clinical threat. The epidemiological survey of Bcc bacteria involved in respiratory infections at the major Portuguese CF Treatment Center at Santa Maria Hospital, in Lisbon, has been carried out by our research group for the past 16 years, covering over 500 clinical isolates where B. cepacia and B. cenocepacia are the predominant species, with B. stabilis, B. contaminans, B. dolosa, and B. multivorans also represented. The systematic and longitudinal study of this CF population during such an extended period of time represents a unique case–study, comprehending 41 Bcc-infected patients (29 pediatric and 12 adult) of whom around 70% have been persistently colonized between 7 months and 9 years. During chronic infection, the CF airways represent an evolving ecosystem, with multiple phenotypic variants emerging from the clonal population and becoming established in the patients’ airways as the result of genetic adaptation. Understanding the evolutionary mechanisms involved is crucial for an improved therapeutic outcome of chronic infections in CF. This review focuses on our contribution to the understanding of these adaptive mechanisms based on extensive phenotypic, genotypic, and genome-wide expression approaches of selected Bcc clonal variants obtained during long-term colonization of the CF airways.

MFS transporters required for multidrug/multixenobiotic (MD/MX) resistance in the model yeast: understanding their physiological function through post-genomic approaches

Multidrug/Multixenobiotic resistance (MDR/MXR) is a widespread phenomenon with clinical, agricultural and biotechnological implications, where MDR/MXR transporters that are presumably able to catalyze the efflux of multiple cytotoxic compounds play a key role in the acquisition of resistance. However, although these proteins have been traditionally considered drug exporters, the physiological function of MDR/MXR transporters and the exact mechanism of their involvement in resistance to cytotoxic compounds are still open to debate. In fact, the wide range of structurally and functionally unrelated substrates that these transporters are presumably able to export has puzzled researchers for years. The discussion has now shifted toward the possibility of at least some MDR/MXR transporters exerting their effect as the result of a natural physiological role in the cell, rather than through the direct export of cytotoxic compounds, while the hypothesis that MDR/MXR transporters may have evolved in nature for other purposes than conferring chemoprotection has been gaining momentum in recent years. This review focuses on the drug transporters of the Major Facilitator Superfamily (MFS; drug:H+ antiporters) in the model yeast Saccharomyces cerevisiae. New insights into the natural roles of these transporters are described and discussed, focusing on the knowledge obtained or suggested by post-genomic research. The new information reviewed here provides clues into the unexpectedly complex roles of these transporters, including a proposed indirect regulation of the stress response machinery and control of membrane potential and/or internal pH, with a special emphasis on a genome-wide view of the regulation and evolution of MDR/MXR-MFS transporters.

Yeast toxicogenomics: lessons from a eukaryotic cell model and cell factory

The yeast Saccharomyces cerevisiae remains a highly relevant experimental model in the field of toxicogenomics and is an important microbial cell factory for the production of added-value chemicals and biofuels. Its deep functional characterization coupled with the straightforward exploitation of Omic approaches and metabolic engineering, at the frontline of systems and synthetic biology, is instrumental to obtain mechanistic insights into the response to multiple toxicants and for the development of robust industrial strains. This critical review focuses on the current field, ranging from the identification of toxicological outcomes of exposure to environmental toxicants, with impact in risk assessment, bioremediation and plant biotechnology, to the improvement of biomass-based biorefinery processes, with applications in pharmacology and in the food and beverages industry.

Proteomic Profiling of Burkholderia cenocepacia Clonal Isolates with Different Virulence Potential Retrieved from a Cystic Fibrosis Patient during Chronic Lung Infection.

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient’s death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.

Genome-Wide Identification of Genes Required for Yeast Growth Under Imatinib Stress: Vacuolar H+-ATPase Function Is an Important Target of This Anticancer Drug

Imatinib is a highly selective tyrosine kinase inhibitor of the oncogenic kinase Bcr-Abl, the result of a chromosomal abnormality that is associated with chronic myeloid leukaemia (CML). Despite the success of this target-directed therapy, imatinib resistance is an emerging problem, especially in advanced stages of CML. In this study, we explored the yeast Saccharomyces cerevisiae as a model eukaryotic system to better understand the mode of action of imatinib, as well as potential mechanisms of resistance to this drug. Using a systematic approach, we screened a yeast haploid deletion collection with individual knockouts of most nonessential yeast genes, and identified 51 genes that are required for yeast resistance to imatinib. The genes identified are involved mainly in DNA repair and transcription control, cell cycle control and differentiation, vacuolar pH homeostasis, vesicular transport, and protein trafficking. Remarkably, approximately 80% of the genes identified in our screen have human orthologs. The vacuolar pH homeostasis function is associated to our dataset by 13 genes that encode subunits and assembly factors of the yeast vacuolar proton-translocating ATPase (V-ATPase). Further studies using fluorescence microscopy showed that physiological acidification of the vacuole is severely compromised following imatinib treatment of yeast cells, an effect that was found to be dose dependent. Results suggest that imatinib might act as an effective inhibitor of V-ATPase function in yeast, identifying V-ATPase activity and vacuolar function as novel imatinib targets.