title: Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.
abstract: Saccharomyces cerevisiae produces antimicrobial peptides (AMPs) during alcoholic fermentation that are active against several wine-related yeasts (e.g. Hanseniaspora guilliermondii) and bacteria (e.g. Oenococcus oeni). In the present study, the physiological changes induced by those AMPs on sensitive H. guilliermondii cells were evaluated in terms of intracellular pH (pHi), membrane permeability and culturability. Membrane permeability was evaluated by staining cells with propidium iodide (PI), pHi was determined by a fluorescence ratio imaging microscopy (FRIM) technique and culturability by a classical plating method. Results showed that the average pHi of H. guilliermondii cells dropped from 6.5 (healthy cells) to 5.4 (damaged cells) after 20 min of exposure to inhibitory concentrations of AMPs, and after 24 h 77.0% of the cells completely lost their pH gradient (∆pH=pHi-pHext). After 24h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1×10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 22.3% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24h, 23% of the AMP-treated cells were sub-lethally injured (with 0<∆pH<3). Taken together, these results indicated that this subpopulation was under a viable but non-culturable (VBNC) state, which was further confirmed by recuperation assays. In summary, our study reveals that these AMPs compromise the plasma membrane integrity (and possibly also the vacuole membrane) of H. guilliermondii cells, disturbing the pHi homeostasis and inducing a loss of culturability.
authors.affiliation: Unit of Bioenergy, Laboratório Nacional de Energia e Geologia (LNEG), Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal; Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Branco
authors.affiliation: Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal; Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark.
authors.forename: Tiago
authors.initials: T
authors.lastname: Viana
authors.affiliation: Unit of Bioenergy, Laboratório Nacional de Energia e Geologia (LNEG), Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal. Electronic address: helena.albergaria@lneg.pt.
authors.forename: Helena
authors.initials: H
authors.lastname: Albergaria
authors.affiliation: Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark.
authors.forename: Nils
authors.initials: N
authors.lastname: Arneborg
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D003593:Cytoplasm / Q000737:chemistry; D005285:Fermentation; D055321:Hanseniaspora / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability / Q000187:drug effects; D011419:Propidium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014920:Wine / Q000382:microbiology
chemical_list: D023181:Antimicrobial Cationic Peptides; D011419:Propidium
keywords: Antimicrobial peptides; Fluorescence ratio imaging microscopy (FRIM); Intracellular pH (pHi); Membrane integrity; Wine yeasts