Publications by authors named "Célia Pais"

51 Publications

Waste-derived volatile fatty acids as carbon source for added-value fermentation approaches.

FEMS Microbiol Lett 2021 05;368(9)

Industrial Biotechnology and Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.

The establishment of a sustainable circular bioeconomy requires the effective material recycling from biomass and biowaste beyond composting/fertilizer or anaerobic digestion/bioenergy. Recently, volatile fatty acids attracted much attention due to their potential application as carbon source for the microbial production of high added-value products. Their low-cost production from different types of wastes through dark fermentation is a key aspect, which will potentially lead to the sustainable production of fuels, materials or chemicals, while diminishing the waste volume. This article reviews the utilization of a volatile fatty acid platform for the microbial production of polyhydroxyalkanoates, single cell oil and omega-3 fatty acids, giving emphasis on the fermentation challenges for the efficient implementation of the bioprocess and how they were addressed. These challenges were addressed through a research project funded by the European Commission under the Horizon 2020 programme entitled 'VOLATILE-Biowaste derived volatile fatty acid platform for biopolymers, bioactive compounds and chemical building blocks'.
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http://dx.doi.org/10.1093/femsle/fnab054DOI Listing
May 2021

Improvement of Genome Annotation: Towards the Exploitation of Genomic Features of a Biotechnologically Relevant Yeast.

J Fungi (Basel) 2021 Apr 10;7(4). Epub 2021 Apr 10.

CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, 4710-057 Braga, Portugal.

is the most commonly used yeast in wine, beer, and bread fermentations. However, 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 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 strains was performed for the first time. eggNOG-mapper was used to perform the functional annotation of the deduced 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 genomes. A comparative study was also conducted between the genome and those from 386 fungal species, revealing a high number of homologous genes with species from the and genera, but also with and . Lastly, the phylogenetic placement of was clarified using the core homologs that were found across 204 common protein sequences of 386 fungal species and strains.
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http://dx.doi.org/10.3390/jof7040287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070057PMC
April 2021

Learning from 80 years of studies: a comprehensive catalogue of non-Saccharomyces yeasts associated with viticulture and winemaking.

FEMS Yeast Res 2021 04;21(3)

CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, 4710-057, Braga, Portugal.

Non-Saccharomyces yeast species are nowadays recognized for their impact on wine´s chemical composition and sensorial properties. In addition, new interest has been given to the commercial exploitation of non-Saccharomyces starter cultures in the wine sector. However, over many years, these yeast species were considered sources of contamination in wine production and conservation, mainly due to the high levels of volatile acidity obtained. The present manuscript systematizes 80 years of literature describing non-Saccharomyces yeast species isolated from grapes and/or grape musts. A link between each reference, the accepted taxonomic name of each species and their geographical occurrence is presented, compiling information for 293 species, in a total of 231 citations. One major focus of this work relates to the isolation of non-Saccharomyces yeasts from grapevines usually ignored in most sampling studies, also as isolation from damaged grapes. These particular niches are sources of specific yeast species, which are not identified in most other explored environments. These yeasts have high potential to be explored for important and diversified biotechnological applications.
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http://dx.doi.org/10.1093/femsyr/foab017DOI Listing
April 2021

Development and Characterization of Monoolein-Based Liposomes of Carvacrol, Cinnamaldehyde, Citral, or Thymol with Anti- Activities.

Antimicrob Agents Chemother 2021 03 18;65(4). Epub 2021 Mar 18.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti- activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of , , , and Finally, the ability of macrophage cells to kill isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 μg/ml of cinnamaldehyde, 256 μg/ml of citral, and 128 μg/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (∼41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages.
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http://dx.doi.org/10.1128/AAC.01628-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097438PMC
March 2021

Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections.

J Fungi (Basel) 2020 Nov 23;6(4). Epub 2020 Nov 23.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710 Braga, Portugal.

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the for the identification of species, and the for the identification of species and . The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.
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http://dx.doi.org/10.3390/jof6040308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712097PMC
November 2020

Single Cell Oil Production by Oleaginous Yeasts Grown in Synthetic and Waste-Derived Volatile Fatty Acids.

Microorganisms 2020 Nov 17;8(11). Epub 2020 Nov 17.

CBMA-Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

Four yeast isolates from the species-, , , and -previously selected by their oleaginous character and growth flexibility in different carbon sources, were tested for their capacity to convert volatile fatty acids into lipids, in the form of single cell oils. Growth, lipid yields, volatile fatty acids consumption, and long-chain fatty acid profiles were evaluated in media supplemented with seven different volatile fatty acids (acetic, butyric, propionic, isobutyric, valeric, isovaleric, and caproic), and also in a dark fermentation effluent filtrate. Yeasts and attained lipid productivities of more than 40% (/), mainly composed of oleic (>40%), palmitic (20%), and stearic (20%) acids, both in synthetic media and in the waste-derived effluent filtrate. These isolates may be potential candidates for single cell oil production in larger scale applications by using alternative carbon sources, combining economic and environmental benefits.
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http://dx.doi.org/10.3390/microorganisms8111809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698568PMC
November 2020

f.a., sp. nov., a novel ascomycetous yeast species isolated from grapes.

Int J Syst Evol Microbiol 2020 Dec;70(12):6307-6312

Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Portugal.

During a study of yeast diversity in Azorean vineyards, four strains were isolated which were found to represent a novel yeast species based on the sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and of the D1/D2 domain of the large subunit (LSU) rRNA gene, together with their physiological characteristics. An additional strain isolated from in Italy had identical D1/D2 sequences and very similar ITS regions (five nucleotide substitutions) to the Azorean strains. Phylogenetic analysis using sequences of the ITS region and D1/D2 domain showed that the five strains are closely related to , although with 56 nucleotide differences in the D2 domain. Intraspecies variation revealed between two and five nucleotide differences, considering the five strains of . Some phenotypic discrepancies support the separation of the new species from their closely related ones, such as the inability to grow at temperatures above 35 °C, to produce acetic acid and the capacity to assimilate starch. Neither conjugations nor ascospore formation were observed in any of the strains. The name f.a., sp. nov., is proposed to accommodate the above noted five strains (holotype, CBS 16465; MycoBank no., MB 835794).
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http://dx.doi.org/10.1099/ijsem.0.004531DOI Listing
December 2020

Oral colonization in healthy individuals: prevalence, genotypic diversity, stability along time and transmissibility.

J Oral Microbiol 2020 Sep 14;12(1):1820292. Epub 2020 Sep 14.

CBMA (Centre of Molecular and Environmental Biology) / Department of Biology / University of Minho, Braga, Portugal.

In this study, 181 healthy individuals, including 29 couples, were analysed regarding oral yeast colonization using a culture-based approach. Results showed that 39% of the individuals were yeast carriers, 89% being colonized with , 5% with , 3% with and 3% with . Sixty-two percent of the couples had at least one member colonized. Colonization and CFU counts were higher in the couples´ group. Eighty percent of the volunteers were colonized with strains with only one CAI genotype, while two but similar CAI genotypes inhabited the oral cavity of the remaining 20% individuals. The same CAI genotypes were found in 66.6% of the couples when both were colonized. Our results indicate that the intimacy among couples increases the probability of heavy cross-colonization, which is potentiated when one member of the couple is a smoker.
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http://dx.doi.org/10.1080/20002297.2020.1820292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534343PMC
September 2020

Population Analysis and Evolution of Mitogenomes.

Microorganisms 2020 Jul 4;8(7). Epub 2020 Jul 4.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057 Braga, Portugal.

The study of mitogenomes allows the unraveling of some paths of yeast evolution that are often not exposed when analyzing the nuclear genome. Although both nuclear and mitochondrial genomes are known to determine phenotypic diversity and fitness, no concordance has yet established between the two, mainly regarding strains' technological uses and/or geographical distribution. In the current work, we proposed a new method to align and analyze yeast mitogenomes, overcoming current difficulties that make it impossible to obtain comparable mitogenomes for a large number of isolates. To this end, 12,016 mitogenomes were considered, and we developed a novel approach consisting of the design of a reference sequence intended to be comparable between all mitogenomes. Subsequently, the population structure of 6646 mitogenomes was assessed. Results revealed the existence of particular clusters associated with the technological use of the strains, in particular regarding clinical isolates, laboratory strains, and yeasts used for wine-associated activities. As far as we know, this is the first time that a positive concordance between nuclear and mitogenomes has been reported for , in terms of strains' technological applications. The results obtained highlighted the importance of including the mtDNA genome in evolutionary analysis, in order to clarify the origin and history of yeast species.
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http://dx.doi.org/10.3390/microorganisms8071001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409325PMC
July 2020

Starmerella vitis f.a., sp. nov., a yeast species isolated from flowers and grapes.

Antonie Van Leeuwenhoek 2020 Sep 24;113(9):1289-1298. Epub 2020 Jun 24.

National Collection of Agricultural and Industrial Microorganisms (NCAIM), Faculty of Food Science, Szent István University, Budapest, Hungary.

A novel yeast species of Starmerella vitis f.a. sp. nov. is proposed to accommodate five strains isolated from flowers, grapes and an insect in the Azores, Canada, Hungary, Palau and Taiwan. As the strains were genetically distinct, we used parsimony network analysis based on ITS-D1/D2 sequences to delineate the species in a statistically objective manner. According to sequence comparisons and phylogenetic analysis, the novel species is most closely related to Starmerella lactis-condensi. The two species cannot be distinguished by conventional physiological tests. The type strain of Starmerella vitis f.a., sp. nov. is CBS 16418; Mycobank number MB 835251.
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http://dx.doi.org/10.1007/s10482-020-01438-xDOI Listing
September 2020

Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source.

BMC Microbiol 2020 03 14;20(1):60. Epub 2020 Mar 14.

CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.

Background: Over the last years oleaginous yeasts have been studied for several energetic, oleochemical, medical and pharmaceutical purposes. However, only a small number of yeasts are known and have been deeply exploited. The search for new isolates with high oleaginous capacity becomes imperative, as well as the use of alternative and ecological carbon sources for yeast growth.

Results: In the present study a high-throughput screening comprising 366 distinct yeast isolates was performed by applying an optimised protocol based on two approaches: (I) yeast cultivation on solid medium using acetic acid as carbon source, (II) neutral lipid estimation by fluorimetry using the lipophilic dye Nile red.

Conclusions: Results showed that, with the proposed methodology, the oleaginous potential of yeasts with broad taxonomic diversity and variety of growth characteristics was discriminated. Furthermore, this work clearly demonstrated the association of the oleaginous yeast character to the strain level, contrarily to the species-level linkage, as usually stated.
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http://dx.doi.org/10.1186/s12866-020-01742-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071767PMC
March 2020

Relevance of Macrophage Extracellular Traps in Killing.

Front Immunol 2019 4;10:2767. Epub 2019 Dec 4.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

causes systemic life-threatening infections, particularly in immunocompromised individuals, such as patients in intensive care units, patients undergoing chemotherapy, and post-surgical and neutropenic patients. The proliferation of invading cells is mainly limited by the action of the human innate immune system, in which phagocytic cells play a fundamental role. This function is, however, limited in neutropenic patients, who rely mainly on the protective immunity mediated by macrophages. Macrophages have been shown to release extracellular DNA fibers, known as macrophage extracellular traps (METs), which can entrap and kill various microbes by a process called ETosis. In this study, we observed that, upon contact with , macrophages became active in phagocyting and engulfing yeast cells. ETosis was induced in 6% of macrophages within the first 30 min of contact, and this percentage increased with the multiplicity of infection until a plateau was reached. After 2.5 h incubation, the presence of extracellular macrophage DNA was observed in approximately half of the cells. This study suggests that the formation of METs occurs before pyroptosis (first 6-8 h) and macrophage cell death (up to 24 h), and thus, METs could be included in models describing -macrophage interactions. We also observed that macrophage ETosis and phagocytosis can occur simultaneously and that, in the first hours of infection, both processes are similarly important in controlling the proliferation of yeast cells, this being critical in neutropenic patients. Finally, it can also be concluded that, since can degrade DNA, the structural component of METs, yeast extracellular DNase activity can be considered as an important virulence factor.
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http://dx.doi.org/10.3389/fimmu.2019.02767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904331PMC
October 2020

Design and validation of a multiplex PCR protocol for microsatellite typing of Candida parapsilosis sensu stricto isolates.

BMC Genomics 2018 Sep 29;19(1):718. Epub 2018 Sep 29.

Laboratorio de Micología Médica, Departamento de Inmunología, Microbiología y Parasitología, UFI11/25 (Microbios y Salud), Facultad de Medicina y Enfermería, Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Apartado 699, E-48080, Bilbao, Spain.

Background: Analysis of polymorphic microsatellite markers (STR) is a helpful genotyping technique to differentiate Candida parapsilosis sensu stricto isolates. The aim of this study is to develop and perform an initial validation of an alternative protocol for the reliable and accurate microsatellite genotyping of C. parapsilosis sensu stricto isolates using high-throughput multiplex PCR. To achieve this, the results obtained using the new protocol were compared to the ones obtained using a previously described reference method. To that end, diagnostic accuracy, informativeness and discrimination parameters were estimated.

Results: Our results showed good concordance between both methods (Kappa index: 0.920), leading to a high sensitivity (1; CI(95%) (0.991-1)) and specificity (1; CI(95%) (0.772-1)) after the validation of the new protocol. Moreover, the electropherograms profiles obtained with the new PCR scheme showed a high signal to noise ratio (SNR).

Conclusions: The new multiplex protocol is valuable for the differentiation of C. parapsilosis sensu stricto, with direct clinical applications. Besides, the new protocol represents a shortening the hands-on time, reducing the sample manipulation (dismissing the possibility of cross-contamination), maintaining the quality of the results (when compared to the ones obtained with the reference method), and helping to the standardization and simplification of the genotyping scheme.
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http://dx.doi.org/10.1186/s12864-018-5065-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162959PMC
September 2018

The Role of Transcription Factor in Response to Carbon Adaptation.

Front Microbiol 2018 29;9:1127. Epub 2018 May 29.

Department of Biology, CBMA, School of Sciences, University of Minho, Braga, Portugal.

is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of transcriptional factor. is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall β-glucans. In addition, absence of has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells.
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http://dx.doi.org/10.3389/fmicb.2018.01127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986929PMC
May 2018

Differentiation of Saccharomyces cerevisiae populations from vineyards of the Azores Archipelago: Geography vs Ecology.

Food Microbiol 2018 Sep 31;74:151-162. Epub 2018 Mar 31.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

Aiming to elucidate the roles that ecology and geography play in shaping the differentiation of fermentative grape-associated Saccharomyces cerevisiae populations, several locations on six islands of the Azores Archipelago were surveyed. A total of 249 strains were isolated from spontaneous fermentations of grape samples from several varieties of two distinct grapevine species (Vitis vinifera L. and Vitis labrusca L.), in vineyards that are under regular cultivation or in abandoned vineyards. Strains were genetically analyzed using a set of nine microsatellite loci, and also phenotypically characterized using relevant physiological/biotechnological tests. Results showed that genetic divergence among populations of the same island was lower than from populations from different islands. Phenotypic comparison of the populations from each of the islands revealed significant differences between them. Strains isolated from the islands with more intensive viticultural activity - Pico, Terceira and Graciosa - showed higher levels of SO tolerance, possibly resulting from selection by human activity. The percentage of strains producing low levels of HS was higher in S. Jorge (60%). Our findings were supported both by genetic and phenotypic data and provide clear evidence for the prevailing role of the geography over ecology in the differentiation of S. cerevisiae populations in the Azores Archipelago.
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http://dx.doi.org/10.1016/j.fm.2018.03.017DOI Listing
September 2018

High variability within Candida albicans transcription factor RLM1: Isolates from vulvovaginal infections show a clear bias toward high molecular weight alleles.

Med Mycol 2018 Jul;56(5):649-651

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.
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http://dx.doi.org/10.1093/mmy/myx079DOI Listing
July 2018

Genomic and transcriptomic analysis of Saccharomyces cerevisiae isolates with focus in succinic acid production.

FEMS Yeast Res 2017 09;17(6)

CBMA (Centre of Molecular and Environmental Biology) / Department of Biology / University of Minho, 4710-057 Braga, Portugal.

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10 selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.
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http://dx.doi.org/10.1093/femsyr/fox057DOI Listing
September 2017

Association between Grape Yeast Communities and the Vineyard Ecosystems.

PLoS One 2017 13;12(1):e0169883. Epub 2017 Jan 13.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Açores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viticultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169883PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234834PMC
August 2017

Yeast Biodiversity in Vineyard Environments Is Increased by Human Intervention.

PLoS One 2016 8;11(8):e0160579. Epub 2016 Aug 8.

Center of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

One hundred and five grape samples were collected during two consecutive years from 33 locations on seven oceanic islands of the Azores Archipelago. Grape samples were obtained from vineyards that were either abandoned or under regular cultivation involving common viticultural interventions, to evaluate the impact of regular human intervention on grape yeast biota diversity in vineyards. A total of 3150 yeast isolates were obtained and 23 yeast species were identified. The predominant species were Hanseniaspora uvarum, Pichia terricola, Starmerella bacillaris and Issatchenkia hanoiensis. The species Barnettozyma californica, Candida azymoides and Pichia cecembensis were reported in grapes or wine-associated environments for the first time. A higher biodiversity was found in active vineyards where regular human intervention takes place (Shannon index: 1.89 and 1.53 in the first and second years, respectively) when compared to the abandoned ones (Shannon index: 0.76 and 0.31). This finding goes against the assumptions that human intervention can destroy biodiversity and lead to homogeneity in the environment. Biodiversity indices were considerably lower in the year with the heaviest rainfall. This study is the first to report on the grape yeast communities from several abandoned vineyards that have undergone no human intervention.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160579PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976982PMC
August 2017

New integrative computational approaches unveil the Saccharomyces cerevisiae pheno-metabolomic fermentative profile and allow strain selection for winemaking.

Food Chem 2016 Nov 13;211:509-20. Epub 2016 May 13.

CBMA - Molecular and Environmental Research Centre, Department of Biology, University of Minho, Braga, Portugal.

During must fermentation by Saccharomyces cerevisiae strains thousands of volatile aroma compounds are formed. The objective of the present work was to adapt computational approaches to analyze pheno-metabolomic diversity of a S. cerevisiae strain collection with different origins. Phenotypic and genetic characterization together with individual must fermentations were performed, and metabolites relevant to aromatic profiles were determined. Experimental results were projected onto a common coordinates system, revealing 17 statistical-relevant multi-dimensional modules, combining sets of most-correlated features of noteworthy biological importance. The present method allowed, as a breakthrough, to combine genetic, phenotypic and metabolomic data, which has not been possible so far due to difficulties in comparing different types of data. Therefore, the proposed computational approach revealed as successful to shed light into the holistic characterization of S. cerevisiae pheno-metabolome in must fermentative conditions. This will allow the identification of combined relevant features with application in selection of good winemaking strains.
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http://dx.doi.org/10.1016/j.foodchem.2016.05.080DOI Listing
November 2016

Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis.

Acta Biomater 2016 07 2;39:133-145. Epub 2016 May 2.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057 Braga, Portugal. Electronic address:

Unlabelled: We evaluated the potential of a liposomal antigen delivery system (ADS) containing Candida albicans cell wall surface proteins (CWSP) in mediating protection against systemic candidiasis. Treatment of bone-marrow-derived dendritic cells with CWSP-loaded dioctadecyldimethylammonium bromide:monoolein (DODAB:MO) liposomes enhanced and prolonged their activation comparatively to free antigen, indicating that liposome-entrapped CWSP were released more sustainable. Therefore, we immunized mice with CWSP either in a free form or loaded into two different DODAB:MO liposome formulations, respectively designated as ADS1 and ADS2, prior to intravenous C. albicans infection. Immunization with ADS1, but not with ADS2, conferred significant protection to infected mice, comparatively to immunization with CWSP or empty liposomes as control. ADS1-immunized mice presented significantly higher serum levels of C. albicans-specific antibodies that enhanced phagocytosis of this fungus. In these mice, a mixed cytokine production profile was observed encompassing IFN-γ, IL-4, IL-17A and IL-10. Nevertheless, only production of IL-4, IL-17 and IL-10 was higher than in controls. In this study we demonstrated that DODAB:MO liposomes enhance the immunogenicity of C. albicans antigens and host protection in a murine model of systemic candidiasis. Therefore, this liposomal adjuvant could be a promising candidate to assess in vaccination against this pathogenic fungus.

Statement Of Significance: This work describes the immunomodulation capacity of the previously validated antigen delivery system (ADS) composed by dioctadecyldimethylammonium bromide (DODAB) and monoolein (MO) lipids incorporating the cell wall surface proteins (CWSP) from C. albicans. Here, we not only present the ability of this system in facilitating antigen uptake by DCs in vitro, but also that this system induces higher levels of pro-inflammatory cytokines and opsonizing specific IgG antibodies in serum of mice immunized subcutaneously. We show that the ADS are efficient nanocarrier and modulate the immune response against intravenous C. albicans infection favoring mouse protection. In sum, we show that the incorporation of C. albicans antigens in DODAB:MO nanocarries are a promising vaccine strategy against C. albicans fungal infection.
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http://dx.doi.org/10.1016/j.actbio.2016.05.001DOI Listing
July 2016

Intrastrain genomic and phenotypic variability of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 reveals microevolutionary adaptation to vineyard environments.

FEMS Yeast Res 2015 Sep 17;15(6). Epub 2015 Jul 17.

CBMA (Centre of Molecular and Environmental Biology)/Department of Biology/University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

The maintenance of microbial species in different environmental conditions is associated with adaptive microevolutionary changes that are shown here to occur within the descendants of the same strain in comparison with the commercial reference strain. However, scarce information is available regarding changes that occur among strain descendants during their persistence in nature. Herein we evaluate genome variations among four isolates of the commercial winemaking strain Saccharomyces cerevisiae Zymaflore VL1 that were re-isolated from vineyards surrounding wineries where this strain was applied during several years, in comparison with the commercial reference strain. Comparative genome hybridization showed amplification of 14 genes among the recovered isolates being related with mitosis, meiosis, lysine biosynthesis, galactose and asparagine catabolism, besides 9 Ty elements. The occurrence of microevolutionary changes was supported by DNA sequencing that revealed 339-427 SNPs and 12-62 indels. Phenotypic screening and metabolic profiles also distinguished the recovered isolates from the reference strain. We herein show that the transition from nutrient-rich musts to nutritionally scarce natural environments induces adaptive responses and microevolutionary changes promoted by Ty elements and by nucleotide polymorphisms that were not detected in the reference strain.
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http://dx.doi.org/10.1093/femsyr/fov063DOI Listing
September 2015

Candida bracarensis: Evaluation of Virulence Factors and its Tolerance to Amphotericin B and Fluconazole.

Mycopathologia 2015 Dec 16;180(5-6):305-15. Epub 2015 Jul 16.

CEB, Centre of Biological Engineering, LIBRO - Laboratório de investigação em Biofilmes Rosário Oliveira, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.
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http://dx.doi.org/10.1007/s11046-015-9925-yDOI Listing
December 2015

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

Med Mycol 2015 May 22;53(4):313-37. Epub 2015 Mar 22.

Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, NSW, Australia.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.
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http://dx.doi.org/10.1093/mmy/myv008DOI Listing
May 2015

Genetic variability of Candida albicans Sap8 propeptide in isolates from different types of infection.

Biomed Res Int 2015 4;2015:148343. Epub 2015 Feb 4.

Biology Department, Minho University, Campus de Gualtar, 4710-057 Braga, Portugal.

The secreted aspartic proteases (Saps) are among the most studied virulence determinants in Candida albicans. These proteins are translated as pre-pro-enzymes consisting of a signal sequence followed by a propeptide and the mature enzyme. The propeptides of secreted proteinases are important for the correct processing, folding/secretion of the mature enzyme. In this study, the DNA sequences of C. albicans Saps were screened and a microsatellite was identified in SAP8 propeptide region. The genetic variability of the repetitive region of Sap8 propeptide was determined in 108 C. albicans independent strains isolated from different types of infection: oral infection (OI), oral commensal (OC), vulvovaginal candidiasis (VVC), and bloodstream infections (BSI). Nine different propeptides for Sap8 processing were identified whose frequencies varied with the type of infection. OC strains presented the highest gene diversity while OI isolated the lowest. The contribution of the Saps to mucosal and systemic infections has been demonstrated and recently Sap8 has been implicated in the cleavage of a signalling glycoprotein that leads to Cek1-MAPK pathway activation. This work is the first to identify a variable microsatellite in the propeptide of a secreted aspartic protease and brings new insights into the variability of Sap8.
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http://dx.doi.org/10.1155/2015/148343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334858PMC
December 2015

Evaluation of T3B fingerprinting for identification of clinical and environmental Sporothrix species.

FEMS Microbiol Lett 2015 Mar 24;362(6). Epub 2015 Feb 24.

Laboratório de Micologia, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ 21045-900, Brazil.

In this study, PCR fingerprinting using the universal primer T3B was applied to distinguish among clinical and environmental species of the Sporothrix complex, Sporothrix brasiliensis, S. globosa, S. mexicana, S. pallida, S. luriei and S. schenckii sensu stricto. The T3B fingerprinting generated clearly distinct banding patterns, allowing the correct identification of all 43 clinical and environmental isolates at the species level, what was confirmed by partial calmodulin gene sequence analyses. This technique is reproducible and provides the identification of all species of the Sporothrix complex with sufficient accuracy to be applied in clinical mycology laboratories as well as in epidemiological studies in order to obtain a better understanding of the epidemiology of sporotrichosis.
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http://dx.doi.org/10.1093/femsle/fnv027DOI Listing
March 2015

Development and optimization of a new MALDI-TOF protocol for identification of the Sporothrix species complex.

Res Microbiol 2015 Feb-Mar;166(2):102-10. Epub 2015 Jan 3.

Laboratório de Micologia, Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.

Accurate species identification of the Sporothrix schenckii complex is essential, since identification based only on phenotypic characteristics is often inconclusive due to phenotypic variability within the species. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification of 70 environmental and clinical isolates of the Sporothrix complex. A reference database was established for MALDI-TOF MS-based species identification according to minor adjustments in the manufacturer's guidelines. The MALDI-TOF MS clearly distinguished strains of Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, S. schenckii, Sporothrix luriei and Sporothrix pallida, enabling identification of all isolates at the species level, as confirmed by partial calmodulin gene sequence analyses. The present methodology is simple, reliable, rapid and highly suitable for routine identification in clinical mycology laboratories and culture collections, particularly for updating and reclassifying of deposited Sporothrix isolates.
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http://dx.doi.org/10.1016/j.resmic.2014.12.008DOI Listing
August 2015

DODAB:monoolein liposomes containing Candida albicans cell wall surface proteins: a novel adjuvant and delivery system.

Eur J Pharm Biopharm 2015 Jan 10;89:190-200. Epub 2014 Dec 10.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal. Electronic address:

We describe the preparation and characterization of DODAB:MO-based liposomes and demonstrate their adjuvant potential and use in antigen delivery. Liposomes loaded with Candida albicans proteins assembled as stable negatively charged spherical nanoparticles with a mean size of 280 nm. High adsorption efficiency (91.0 ± 9.0%) is attained with high lipid concentrations. The nanoparticles were non-toxic, avidly taken up by macrophage cells and accumulated in membrane rich regions with an internalization time of 20 min. Immunized mice displayed strong humoral and cell-mediated immune responses, producing antibodies (IgGs) against specific cell wall proteins, Cht3p and Xog1p. DODAB:MO-based liposomes loaded with C. albicans proteins have an excellent immunogenic potential and can be explored for the development of an immunoprotective strategy against Candida infections.
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http://dx.doi.org/10.1016/j.ejpb.2014.11.028DOI Listing
January 2015

Different scenarios for Candida parapsilosis fungaemia reveal high numbers of mixed C. parapsilosis and Candida orthopsilosis infections.

J Med Microbiol 2015 Jan 28;64(Pt 1):7-17. Epub 2014 Oct 28.

Laboratório de Micologia, Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.

Nosocomial fungal bloodstream infections (BSI) are increasing significantly in hospitalized patients and Candida parapsilosis has emerged as an important pathogen responsible for numerous outbreaks. The objective of this study was to evaluate C. parapsilosis sensu lato infection scenarios, regarding species distribution and strain relatedness. One hundred isolates of C. parapsilosis sensu lato derived from blood cultures and catheter tips were analysed by multiplex microsatellite typing and by sequencing D1/D2 regions of the ribosomal DNA. Our results indicate that 9.5 % of patients presented infections due to C. parapsilosis and Candida orthopsilosis, 57.1 % due to C. parapsilosis, 28.3 % due to C. orthopsilosis and 4.8 % due to Candida metapsilosis. Eighty per cent of the C. parapsilosis BSIs were due to a single strain that was also identified in the catheter, but in 10 % of the cases C. parasilosis was identified in the catheter but the BSI was due to C. orthopsilosis. There is a significant probability that C. parapsilosis isolates collected from the same patient at more than 3 months interval are of different strains (P = 0.0179). Moreover, several isolates were identified persistently in the same hospital, infecting six different patients. The incidence of polyfungal BSI infections with C. parapsilosis and C. orthopsilosis is reported herein for the first time, emphasizing the fact that the species identified in the catheter is not always responsible for the BSI, thus impacting the treatment strategy. The observation that strains can remain in the hospital environment for years highlights the possible existence of reservoirs and reinforces the need for accurate genotyping tools, such as the markers used for elucidating epidemiological associations and detecting outbreaks.
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http://dx.doi.org/10.1099/jmm.0.080655-0DOI Listing
January 2015
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