Browsing by Author "Arneborg, Nils"
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- Antimicrobial peptides from Saccharomyces cerevisiae induce physiological changes in Hanseniaspora guilliermondiiPublication . Branco, Patricia; Albergaria, Helena; Arneborg, NilsSaccharomyces cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against other wine-related yeasts (e.g. Hanseniaspora guilliermondii) (Albergaria et al., 2010) and bacteria (e.g. Oenococcus oeni) (Osborne and Edwards, 2007). In the present study we assessed the physiological changes induced by those AMPs on sensitive yeast cells of Hanseniaspora guilliermondii, namely membrane permeability and intracellular pH (pHi) alterations. Membrane permeability was evaluated by staining cells with propidium iodide (PI) and pHi by the fluorescence ratio imaging microscopy (FRIM) technique (Guldfeldt and Arneborg, 1998). Results showed that after 20 min of incubation with inhibitory concentrations of AMPs, the average pHi of cells dropped from 6.5 to 5.4. After 8 h of incubation, 32% of the cells had lost their ĢpH (=pHi-pHext) and after 24 h that percentage rose to 77%. The culturability (plating) and viability (PI staining) of the sensitive yeast cells also decreased in the presence of the AMPs. After 24 h of exposure to AMPs, 61% of the cells were dead (PI-stained) and the number of viable cells fell from 1 ~105 to 1.5 CFU/ml, which means that virtually all cells (99.999%) became unculturable but a sub-population of 39% of cells remained in a viable but non-culturable (VBNC) state. However, those VBNC cells were able to recover their culturability after incubation at optimal growth conditions. Our study revealed that the mode of action of these AMPs seems to be primarily targeted to the cell membranes, reducing their permeability and preventing cells to maintain pH homeostasis.
- Dominance of Saccharomyces cerevisiae in wine fermentations: secretion of antimicrobial peptides and microbial interactionsPublication . Albergaria, Helena; Branco, Patricia; Francisco, Diana; Coutinho, Rute; Monteiro, Margarida; Malfeito-Ferreira, Manuel; Arneborg, Nils; Almeida, M. Gabriela; Caldeira, JorgeIn present work we investigated the antagonistic effect by S. cerevisiae against several wine-related microbial species vis-à-vis the secretion of antimicrobial peptides (AMPs).
- Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux ratesPublication . Branco, Patricia; Albergaria, Helena; Arneborg, Nils; Prista, CatarinaABSTRACT: Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.
- Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactionsPublication . Branco, Patricia; Francisco, Diana; Chambon, Christophe; Hébraud, Michel; Arneborg, Nils; Gabriela Almeida, M.; Caldeira, Jorge; Albergaria, HelenaSaccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vastworldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.
- Interactions between Saccharomyces cerevisiae and Hanseniaspora guilliermondii: cell-cell contact mechanismPublication . Branco, Patricia; Kemsawasd, Varongsiri; Arneborg, Nils; Albergaria, HelenaSeveral studies have shown that the early death of non-Saccharomyces during wine fermentations are due to yeast-yeast interactions induced by Saccharomyces cerevisiae (Sc) through different mechanisms such as growth arrest mediated by a cell-cell contact mechanism (Nissen et al. 2003) and death mediated by killer-like toxins (Pérez-Nevado et al 2006; Albergaria et al. 2010). Besides, previous work also showed that death of non-Saccharomyces in co-cultivation with Sc is always triggered at the end of exponential growth phase (Pérez-Nevado et al 2006). In order to investigate the role of cell-cell contact in the early death of non-Saccharomyces, we performed assays in which Sc cells pre-grown at enological growth conditions for 12 and 48 h, respectively, were in direct contact with Hanseniaspora guilliermondii (Hg) cells at high cellular density (107-108 cells/ml) in a carbon-free medium. As a negative control we performed similar assays in which Sc and Hg cells were separated by a dialysis tube (pore cut-off of 1000 kDa) and as a positive control a single Hg culture. Results showed that Hg cell density decreased by 2 orders of magnitude (i.e. from 108-106 cells/ml) in contact with 48 h-grown Sc cells, while its viability remained unchanged (108cfu/ml) in the presence of 12 h-grown Sc cells. Moreover, Hg viability was not affected both in the dialysis tube experiments and single culture, which confirmed the death-induced cell-cell contact phenomenon.
- Proteomics insights into the responses of Saccharomyces cerevisiae during mixed-culture alcoholic fermentation with Lachancea thermotoleransPublication . Peng, Chuantao; Andersen, Birgit; Arshid, Samina; Larsen, Martin R.; Albergaria, Helena; Lametsch, Rene; Arneborg, NilsABSTRACT: The response of Saccharomyces cerevisiae to cocultivation with Lachancea thermotolerans during alcoholic fermentations has been investigated using tandem mass tag (TMT)-based proteomics. At two key time-points, S. cerevisiae was sorted from single S. cerevisiae fermentations and from mixed fermentations using flow cytometry sorting. Results showed that the purity of sorted S. cerevisiae was above 96% throughout the whole mixed-culture fermentation, thereby validating our sorting methodology. By comparing protein expression of S. cerevisiae with and without L. thermotolerans, 26 proteins were identified as significantly regulated proteins at the early death phase (T1), and 32 significantly regulated proteins were identified at the late death phase (T2) of L. thermotolerans in mixed cultures. At T1, proteins involved in endocytosis, increasing nutrient availability, cell rescue and resistance to stresses were upregulated, and proteins involved in proline synthesis and apoptosis were downregulated. At T2, proteins involved in protein synthesis and stress responses were up- and downregulated, respectively. These data indicate that S. cerevisiae was stressed by the presence of L. thermotolerans at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from stress, perhaps increasing its enzymatic machinery to ensure better survival.