Saccharomycin, a biocide from S. cerevisiae that kill-off other yeasts

Abstract

Introduction: Saccharomyces cerevisiae plays an important role in alcoholic fermentation and is involved in the production of wine, beer and bread. Recent studies [1–7] showed that S. cerevisiae secretes antimicrobial peptides (AMPs), named “saccharomycin”, derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against a variety of wine-related microbial species. AMPs are low molecular weight proteins with broad antimicrobial spectrum of action against bacteria, viruses, and fungi [8]. Organisms use AMPs for defence against infection and membrane interaction appears to be the key to this antimicrobial function: generally they adopt amphiphilic structures that interact with the infectious agent’s membrane. AMPs constitute a promising source as alternatives to: i) combat pathogenic bacteria resistant to common antibiotics and ii) substitute chemical preservatives in food-fermented products such as wine. The aim of this study was to discover the mode of action of these peptides by detailed chemical structure characterisation and cell contact mechanism.

Materials and methods: Structural characterization of saccharomycin and of its synthetic analogues was performed by NMR. 2D NMR spectra, COSY, TOCSY, ROESY, and 1H-13C-HMQC (Figure 1) for identifying non-peptide components involved in the chemical structure of saccharomycin by comparison with synthetic analogues. Atomic Force Microscopy (Figure 2) was used to observe the surface of different species of yeast cells upon cell-cell contact. Yeast cells were observed after being collected at different growth stages, either from mixed or pure cultures. Differential proteomic studies were performed to identify S.cerevisiae metabolic pathways involved in the production of AMPs using 2D electrophoresis coupled to MS techniques

Figure 1. ¹³C HMQC of native AMPs from S. cerevisiae

Figure 2. ¹³AFM image of S. cerevisiae cell surface.

Results: NMR experiments show further support of our previous results indicating the presence of carbohydrate molecules linked to the native peptide. AFM images of pure and mixed cultures were obtained in different growth times, at different dilutions.

Discussion and conclusions: AFM imaging shows direct cell-cell contact between different yeast species in mixed cultures. Protemic studies have identified main proteins in this methabolism. 2D NMR spectra of biological active isolated fraction from S. cerevisiae gave evidence of the presence of glycosylated peptides in the native form.

Acknowledgements

Financed by FCT: PTDC/AGR-ALI/113565/2009 BIOPEPTIDES- Biopreservation of Ethanoic Fermentations: antimicrobial activity, biochemical properties and molecular characterization of yeast peptides. FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728)