FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

Abstract

bSaccharomyces cerevisiae "flor" yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends on FLO11 gene length and expression. FLO11 encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm of S. cerevisiae flor strains. We found that PAF26, a synthetic tryptophanrich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functional FLO11 but was not accompanied by increased FLO11 expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that a flo11 gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that the FLO11 gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-tocell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.

Interactions by the Fungal Flo11 Adhesin Depend on a Fibronectin Type III-like Adhesin Domain Girdled by Aromatic Bands

“…Tryptophan was suggested previously to be somehow involved in FLO11-mediated hydrophobic interactions between S. cerevisiae cells and polystyrene surfaces (Mortensen et al, 2007). Likewise, the hydrophobic PAF26 peptide that promotes FLO11-dependent biofilm formation is enriched with tryptophans (Bou Zeidan et al, 2013). The aromatic bands exposed by the ScFlo11A domain now rationalize these observations.…”

“…Moreover, yeast cells in biofilms adhere not only to each other, but also to substrate surfaces such as agar or plastic surfaces (Mortensen et al, 2007). This ability of Flo11 was attributed to its general hydrophobic characteristics (Reynolds and Fink, 2001) and can be further enhanced by a small hydrophobic and basic peptide, PAF26 (RKKWFW), which apparently acts as a bridge in Flo11-mediated cell interactions (Bou Zeidan et al, 2013). FLO11 is also required for the formation of fibrous interconnections between cells of colony-grown S. cerevisiae wild strains (Vachova et al, 2011).…”

The Penicillium digitatum protein O‐mannosyltransferase Pmt2 is required for cell wall integrity, conidiogenesis, virulence and sensitivity to the antifungal peptide PAF26

“…Glycan patterns from CW proteins have been proposed to act as docking moieties for the binding (and therefore activity) of antifungal peptides (Harris et al ., ; Marcos and Gandía, ). The deletion of the glycosylated highly abundant CW protein Flo11 abolished the binding of PAF26 to wine ‘flor’ strains of yeast, which supports this view (Bou Zeidan et al ., ). The glycosylated domain of Msb2 from C. albicans is capable of binding to the antifungal peptides histatin 5 and LL‐37, and its release into the medium protects cells by sequestration of the peptide (Szafranski‐Schneider et al ., ).…”

Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides

“…Although the precise antibacterial mechanism of concatemeric PAFs needs to be studied in detail, their lack of hemolytic activity indicates that they do not act simply by membrane permeabilization. In the case of filamentous fungi and yeast, the activity of PAF26 is mediated by the initial interaction with the fungal cell wall and cell wall glycoproteins (Bou Zeidan et al 2013;Harries et al 2013). Therefore, the role of the bacterial cell wall in the activity of PAF concatemers should be examined in the future for the characterization of the antibacterial activity.…”