RESUMO
Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.
Assuntos
Hanseniaspora , Vinho , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Hanseniaspora/genética , Vinho/análise , Ésteres/análise , Filogenia , Fermentação , Álcoois/metabolismo , Acetatos/metabolismo , Nitrogênio/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismoRESUMO
BACKGROUND: Saccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO2). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome. RESULTS: Resequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall ß-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO2-tolerance genes, were identified in S. ludwigii genome. CONCLUSIONS: This work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming.
Assuntos
Hanseniaspora , Vinho , Fermentação , Saccharomyces cerevisiae , SaccharomycetalesRESUMO
BACKGROUND: In this paper, we explore the concept of multi-objective optimization in the field of metabolic engineering when both continuous and integer decision variables are involved in the model. In particular, we propose a multi-objective model that may be used to suggest reaction deletions that maximize and/or minimize several functions simultaneously. The applications may include, among others, the concurrent maximization of a bioproduct and of biomass, or maximization of a bioproduct while minimizing the formation of a given by-product, two common requirements in microbial metabolic engineering. RESULTS: Production of ethanol by the widely used cell factory Saccharomyces cerevisiae was adopted as a case study to demonstrate the usefulness of the proposed approach in identifying genetic manipulations that improve productivity and yield of this economically highly relevant bioproduct. We did an in vivo validation and we could show that some of the predicted deletions exhibit increased ethanol levels in comparison with the wild-type strain. CONCLUSIONS: The multi-objective programming framework we developed, called MOMO, is open-source and uses POLYSCIP (Available at http://polyscip.zib.de/). as underlying multi-objective solver. MOMO is available at http://momo-sysbio.gforge.inria.fr.
Assuntos
Engenharia Metabólica/métodos , Software , Biomassa , Etanol/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Farmacorresistência Fúngica/genética , Candida glabrata/isolamento & purificação , Clotrimazol/farmacologia , Ergosterol/metabolismo , Fluconazol/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Fatores de Transcrição/genética , Transcriptoma/genética , Triazóis/farmacologia , Voriconazol/farmacologiaRESUMO
The mechanisms of persistence and virulence associated with Candida glabrata infections are poorly understood, limiting the ability to fight this fungal pathogen. In this study, the multidrug resistance transporters CgTpo1_1 and CgTpo1_2 are shown to play a role in C. glabrata virulence. The survival of the infection model Galleria mellonella, infected with C. glabrata, was found to increase upon the deletion of either CgTPO1_1 or CgTPO1_2. The underlying mechanisms were further explored. In the case of CgTpo1_1, this phenotype was found to be consistent with the observation that it confers resistance to antimicrobial peptides (AMP), such as the human AMP histatin-5. The deletion of CgTPO1_2, on the other hand, was found to limit the survival of C. glabrata cells when exposed to phagocytosis and impair biofilm formation. Interestingly, CgTPO1_2 expression was found to be up-regulated during biofilm formation, but and its deletion leads to a decreased expression of adhesin-encoding genes during biofilm formation, which is consistent with a role in biofilm formation. CgTPO1_2 expression was further seen to decrease plasma membrane potential and affect ergosterol and fatty acid content. Altogether, CgTpo1_1 and CgTpo1_2 appear to play an important role in the virulence of C. glabrata infections, being at the cross-road between multidrug resistance and pathogenesis.
Assuntos
Biofilmes , Candida glabrata/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Animais , Antifúngicos/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/patogenicidade , Resistência a Múltiplos Medicamentos , Ergosterol/metabolismo , Ácidos Graxos/metabolismo , Expressão Gênica , Genes Fúngicos , Hemócitos/microbiologia , Histatinas/farmacologia , Humanos , Larva/microbiologia , Metabolismo dos Lipídeos , Potenciais da Membrana , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Mariposas , Fagocitose , VirulênciaRESUMO
The action of benzoic acid in the food and beverage industries is compromised by the ability of spoilage yeasts to cope with this food preservative. Benzoic acid occurs naturally in many plants and is an intermediate compound in the biosynthesis of many secondary metabolites. The understanding of the mechanisms underlying the response and resistance to benzoic acid stress in the eukaryotic model yeast is thus crucial to design more suitable strategies to deal with this toxic lipophilic weak acid. In this study, the Saccharomyces cerevisiae multidrug transporter Tpo1 was demonstrated to confer resistance to benzoic acid. TPO1 transcript levels were shown to be up-regulated in yeast cells suddenly exposed to this stress agent. This up-regulation is under the control of the Gcn4 and Stp1 transcription factors, involved in the response to amino acid availability, but not under the regulation of the multidrug resistance transcription factors Pdr1 and Pdr3 that have binding sites in TPO1 promoter region. Benzoic acid stress was further shown to affect the intracellular pool of amino acids and polyamines. The observed decrease in the concentration of these nitrogenous compounds, registered upon benzoic acid stress exposure, was not found to be dependent on Tpo1, although the limitation of yeast cells on nitrogenous compounds was found to activate Tpo1 expression. Altogether, the results described in this study suggest that Tpo1 is one of the key players standing in the crossroad between benzoic acid stress response and tolerance and the control of the intracellular concentration of nitrogenous compounds. Also, results can be useful to guide the design of more efficient preservation strategies and the biotechnological synthesis of benzoic acid or benzoic acid-derived compounds.
Assuntos
Antiporters/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ácido Benzoico/farmacologia , Proteínas Nucleares/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Aminoácidos , Antiporters/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Farmacorresistência Fúngica Múltipla/genética , Tolerância a Medicamentos , Conservantes de Alimentos , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Poliaminas , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores , Fatores de Transcrição/genética , Ativação Transcricional , Regulação para CimaRESUMO
BACKGROUND: The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S. cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniaspora guilliermondii, this being the first time that this relevant yeast-yeast interaction is examined at a genomic scale. RESULTS: Co-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds. CONCLUSIONS: By integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation.
Assuntos
Fermentação , Hanseniaspora/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho , Técnicas de Cocultura , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genômica , Interações Microbianas , Saccharomyces cerevisiae/genéticaRESUMO
OBJECTIVES: The ability of opportunistic pathogenic Candida species to persist and invade specific niches in the human host depends on their resistance to natural growth inhibitors and antifungal therapy. This work describes the role of the Candida glabrata drug:H(+) antiporter CgTpo3 (ORF CAGL0I10384g) in this context. METHODS: Deletion and cloning of CgTPO3 was achieved using molecular biology tools. C. glabrata strain susceptibility was assayed based on growth in liquid and solid media and through MIC determination. Radiolabelled compound accumulation or HPLC were used for the assessment of the role of CgTpo3 as a drug or polyamine transporter. Quantitative RT-PCR was used for expression analysis. RESULTS: CgTpo3 was found to confer resistance to azole drugs in C. glabrata. This protein was found to be localized to the plasma membrane and to decrease the intracellular accumulation of [(3)H]clotrimazole, playing a direct role in its extrusion from pre-loaded C. glabrata cells. CgTPO3 was further found to confer resistance to spermine, complementing the susceptibility phenotypes exhibited by the deletion of its Saccharomyces cerevisiae homologue, TPO3. In spermine-stressed C. glabrata cells, CgTPO3 is transcriptionally activated in a CgPdr1-dependent manner, contributing to a decrease in the intracellular concentration of this polyamine. Clotrimazole exposure was found to lead to the intracellular accumulation of spermine, and pre-exposure to this polyamine was found consistently to lead to increased clotrimazole resistance. CONCLUSIONS: Altogether, these results point to a significant role for CgTpo3 in azole drug resistance and in the tolerance to high polyamine concentrations, such as those found in the urogenital tract.
Assuntos
Antiporters/metabolismo , Azóis/metabolismo , Candida glabrata/metabolismo , Farmacorresistência Fúngica , Poliaminas/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Deleção de Genes , Perfilação da Expressão Gênica , Homeostase , Marcação por Isótopo , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A novel quartz crystal microbalance (QCM) analytical method is developed based on the transmission line model (TLM) algorithm to analyze the binding of transcription factors (TFs) to immobilized DNA oligoduplexes. The method is used to characterize the mechanical properties of biological films through the estimation of the film dynamic shear moduli, G and G, and the film thickness. Using the Saccharomyces cerevisiae transcription factor Haa1 (Haa1DBD) as a biological model two sensors were prepared by immobilizing DNA oligoduplexes, one containing the Haa1 recognition element (HRE(wt)) and another with a random sequence (HRE(neg)) used as a negative control. The immobilization of DNA oligoduplexes was followed in real time and we show that DNA strands initially adsorb with low or non-tilting, laying flat close to the surface, which then lift-off the surface leading to final film tilting angles of 62.9° and 46.7° for HRE(wt) and HRE(neg), respectively. Furthermore we show that the binding of Haa1DBD to HRE(wt) leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HRE(wt) oligoduplex. This work demonstrates the suitability of the QCM to monitor the specific binding of TFs to immobilized DNA sequences and provides an analytical methodology to study protein-DNA biophysics and kinetics.
Assuntos
DNA/química , Modelos Teóricos , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , DNA/metabolismo , Ligação Proteica , Quartzo , Saccharomyces cerevisiae/químicaRESUMO
Advanced impedance spectroscopy analysis based on the transmission line model (TLM) is explored as a novel QCM acoustic biosensing platform for the detection of the single point mutation effect on the binding of the transcription factors (TFs) to immobilized DNA oligoduplexes and the characterization of the protein-DNA mechanical properties.
Assuntos
Espectroscopia Dielétrica/métodos , Ácidos Nucleicos Imobilizados/metabolismo , Mutação Puntual , Técnicas de Microbalança de Cristal de Quartzo/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ácidos Nucleicos Imobilizados/química , Modelos Moleculares , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/químicaRESUMO
The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.
Assuntos
Genes Fúngicos , Microbiologia Industrial , Lignina/metabolismo , Saccharomyces cerevisiae/genética , Ácido Acético/toxicidade , Biomassa , Farmacorresistência Fúngica , Fermentação , Furaldeído/toxicidade , Genoma Fúngico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , TriticumRESUMO
Vaginal infectious diseases caused by viruses and bacteria have been linked to the occurrence of dysbiosis, that is, a reduction in the abundance of the normally dominating vaginal Lactobacillus species. Mucosal infections in the vagina and/or vulva caused by Candida species, usually known as vulvovaginal candidiasis (or VVC), are among the leading causes of diseases in the vaginal tract. The existence of a clear link between the occurrence of dysbiosis and the development of VVC is still unclear, although multiple observations point in that direction. Based on the idea that vaginal health is linked to a microbiota dominated by lactobacilli, several probiotics have been used in management of VVC, either alone or in combination with antifungals, having obtained different degrees of success. In most cases, the undertaken trials resorted to lactobacilli species other than those indigenous to the vaginal tract, although in vitro these vaginal species were shown to reduce growth, viability and virulence of Candida. In this paper we overview the role of lactobacilli and Candida in the vaginal micro- and myco-biomes, while discussing the results obtained in what concerns the establishment of interference mechanisms in vivo and the environmental factors that could determine that. We also overview the molecular mechanisms by which lactobacilli species have been shown to inhibit pathophysiology of Candida, including the description of the genes and pathways determining their ability to thrive in the presence of each other. In a time where concerns are increasing with the emergence of antifungal resistance and the slow pace of discovery of new antifungals, a thorough understanding of the molecular mechanisms underneath the anti-Candida effect prompted by vaginal lactobacilli is of utmost importance to assure a knowledge-based design of what can be a new generation of pharmaceuticals, eventually focusing therapeutic targets other than the usual ones.
Assuntos
Candida , Candidíase Vulvovaginal , Feminino , Humanos , Lactobacillus/genética , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Disbiose , Vagina/microbiologia , Candidíase Vulvovaginal/microbiologia , Candidíase Vulvovaginal/terapia , Candida albicansRESUMO
The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5'-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3'. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (K(D) of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (K(D) of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (K(D) of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5'-(G/C)(A/C)GG(G/C)G-3'. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed.
Assuntos
Ácido Acético/toxicidade , Regulação Fúngica da Expressão Gênica , Elementos de Resposta , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA Fúngico/química , Redes Reguladoras de Genes , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Ativação TranscricionalRESUMO
The exploration of the interference prompted by commensal bacteria over fungal pathogens is an interesting alternative to develop new therapies. In this work we scrutinized how the presence of the poorly studied vaginal species Lactobacillus gasseri affects relevant pathophysiological traits of Candida albicans and Candida glabrata. L. gasseri was found to form mixed biofilms with C. albicans and C. glabrata resulting in pronounced death of the yeast cells, while bacterial viability was not affected. Reduced viability of the two yeasts was also observed upon co-cultivation with L. gasseri under planktonic conditions. Either in planktonic cultures or in biofilms, the anti-Candida effect of L. gasseri was augmented by acetate in a concentration-dependent manner. During planktonic co-cultivation the two Candida species counteracted the acidification prompted by L. gasseri thus impacting the balance between dissociated and undissociated organic acids. This feature couldn't be phenocopied in single-cultures of L. gasseri resulting in a broth enriched in acetic acid, while in the co-culture the non-toxic acetate prevailed. Altogether the results herein described advance the design of new anti-Candida therapies based on probiotics, in particular, those based on vaginal lactobacilli species, helping to reduce the significant burden that infections caused by Candida have today in human health.
RESUMO
Zygosaccharomyces bailii is the most tolerant yeast species to acetic acid-induced toxicity, being able to grow in the presence of concentrations of this food preservative close to the legal limits. For this reason, Z. bailii is the most important microbial contaminant of acidic food products but the mechanisms behind this intrinsic resistance to acetic acid are very poorly characterized. To gain insights into the adaptive response and tolerance to acetic acid in Z. bailii, we explored an expression proteomics approach, based on quantitative 2DE, to identify alterations occurring in the protein content in response to sudden exposure or balanced growth in the presence of an inhibitory but nonlethal concentration of this weak acid. A coordinate increase in the content of proteins involved in cellular metabolism, in particular, in carbohydrate metabolism (Mdh1p, Aco1p, Cit1p, Idh2p, and Lpd1p) and energy generation (Atp1p and Atp2p), as well as in general and oxidative stress response (Sod2p, Dak2p, Omp2p) was registered. Results reinforce the concept that glucose and acetic acid are coconsumed in Z. bailii, with acetate being channeled into the tricarboxylic acid cycle. When acetic acid is the sole carbon source, results suggest the activation of gluconeogenic and pentose phosphate pathways, based on the increased content of several proteins of these pathways after glucose exhaustion.
Assuntos
Ácido Acético/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteoma/efeitos dos fármacos , Zygosaccharomyces/efeitos dos fármacos , Zygosaccharomyces/fisiologia , Análise de Variância , Metabolismo dos Carboidratos , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/análise , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Redes e Vias Metabólicas , Estresse Oxidativo/fisiologia , Proteoma/metabolismo , Proteômica , Zygosaccharomyces/metabolismoRESUMO
MOTIVATION: Uncovering mechanisms underlying gene expression control is crucial to understand complex cellular responses. Studies in gene regulation often aim to identify regulatory players involved in a biological process of interest, either transcription factors coregulating a set of target genes or genes eventually controlled by a set of regulators. These are frequently prioritized with respect to a context-specific relevance score. Current approaches rely on relevance measures accounting exclusively for direct transcription factor-target interactions, namely overrepresentation of binding sites or target ratios. Gene regulation has, however, intricate behavior with overlapping, indirect effect that should not be neglected. In addition, the rapid accumulation of regulatory data already enables the prediction of large-scale networks suitable for higher level exploration by methods based on graph theory. A paradigm shift is thus emerging, where isolated and constrained analyses will likely be replaced by whole-network, systemic-aware strategies. RESULTS: We present TFRank, a graph-based framework to prioritize regulatory players involved in transcriptional responses within the regulatory network of an organism, whereby every regulatory path containing genes of interest is explored and incorporated into the analysis. TFRank selected important regulators of yeast adaptation to stress induced by quinine and acetic acid, which were missed by a direct effect approach. Notably, they reportedly confer resistance toward the chemicals. In a preliminary study in human, TFRank unveiled regulators involved in breast tumor growth and metastasis when applied to genes whose expression signatures correlated with short interval to metastasis.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ácido Acético/farmacologia , Sítios de Ligação , Humanos , Metástase Neoplásica , Quinina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. RESULTS: A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased ³H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. CONCLUSIONS: PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Etanol/metabolismo , Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação para Cima , Transportadores de Cassetes de Ligação de ATP/genética , Fermentação , Regulação Fúngica da Expressão Gênica , Hipergravidade , Microbiologia Industrial , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The pathogenic yeast Candida glabrata is intrinsically resilient to azoles and rapidly acquires resistance to these antifungals, in vitro and in vivo. In most cases azole-resistant C. glabrata clinical strains encode hyperactive CgPdr1 variants, however, resistant strains encoding wild-type CgPDR1 alleles have also been isolated, although remaining to be disclosed the underlying resistance mechanism. In this study, we scrutinized the mechanisms underlying resistance to azoles of 8 resistant clinical C. glabrata strains, identified along the course of epidemiological surveys undertaken in Portugal. Seven of the strains were found to encode CgPdr1 gain-of-function variants (I392M, E555K, G558C, and I803T) with the substitutions I392M and I803T being herein characterized as hyper-activating mutations for the first time. While cells expressing the wild-type CgPDR1 allele required the mediator subunit Gal11A to enhance tolerance to fluconazole, this was dispensable for cells expressing the I803T variant indicating that the CgPdr1 interactome is shaped by different gain-of-function substitutions. Genomic and transcriptomic profiling of the sole azole-resistant C. glabrata isolate encoding a wild-type CgPDR1 allele (ISTB218) revealed that under fluconazole stress this strain over-expresses various genes described to provide protection against this antifungal, while also showing reduced expression of genes described to increase sensitivity to these drugs. The overall role in driving the azole-resistance phenotype of the ISTB218 C. glabrata isolate played by these changes in the transcriptome and genome of the ISTB218 isolate are discussed shedding light into mechanisms of resistance that go beyond the CgPdr1-signalling pathway and that may alone, or in combination, pave the way for the acquisition of resistance to azoles in vivo.
Assuntos
Azóis , Candida glabrata , Alelos , Antifúngicos/farmacologia , Azóis/farmacologia , Farmacorresistência Fúngica/genética , Fluconazol/farmacologia , Mutação com Ganho de Função , Regulação Fúngica da Expressão Gênica , Genômica , Testes de Sensibilidade Microbiana , TranscriptomaRESUMO
Currently there is a gap between the rate of new antifungal development and the emergence of resistance among Candida clinical strains, particularly threatened by the extreme adhesiveness of C. albicans to indwelling medical devices. Two silver camphorimine complexes, [Ag(OH){OC10H14N(C6H4)2NC10H14O}] (compound P) and [{Ag(OC10H14NC6H4CH3-p)}2(µ-O)] (compound Q), are herein demonstrated as having high inhibiting activity towards the growth of Candida albicans and Candida glabrata clinical strains resistant to azoles, the frontline antifungals used in clinical practice. Compounds P and Q were also explored as bioactive coatings to prevent colonization by C. albicans and colonize the surface of indwelling medical devices, resulting in persistent infections. Functionalization of stainless steel with polycaprolactone (PCL) matrix embedded with compounds P or Q was reported for the first time to inhibit the colonization of C. albicans by 82% and 75%, respectively. The coating of PCL loaded with Q or P did not cause cytotoxic effects in mammalian cells, demonstrating the biocompatibility of the explored approach. The identification and further exploration of new approaches for surface engineering based on new molecules that can sensitize resistant strains, as herein demonstrated for complexes P and Q, is a significant step forward to improve the successful treatment of candidiasis.
RESUMO
Microbially produced carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals. Most CAs are intermediates of microbial central carbon metabolism, and therefore, a biochemical production pathway is described and can be transferred to a host of choice to enable/improve production at an industrial scale. However, for some CAs, the implementation of this approach is difficult, either because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). Synthetic biology has been reshaping the range of molecules that can be produced by microbial cells by setting new-to-nature pathways that leverage on enzyme arrangements not observed in vivo, often in association with the use of substrates that are not enzymes' natural ones. In this review, we provide an overview of how the establishment of synthetic pathways, assisted by computational tools for metabolic retrobiosynthesis, has been applied to the field of CA production. The translation of these efforts in bridging the gap between the synthesis of CAs and of their more interesting derivatives, often themselves non-naturally occurring molecules, is also reviewed using as case studies the production of methacrylic, methylmethacrylic and poly-lactic acids.