Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells

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 (Delta pH = pHi - pH(ext)). After 24 h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1 x 10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 223% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24 h, 23% of the AMP-treated cells were sub-lethally injured (with 0 < Delta 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. (C) 2015 Elsevier B.V. All rights reserved.