The resistance of the yeast Saccharomyces cerevisiae to the biocide polyhexamethylene biguanide: involvement of cell wall integrity pathway and emerging role for YAP1

Abstract

Background: Polyhexamethylene biguanide (PHMB) is an antiseptic polymer that is mainly used for cleaning hospitals and pools and combating Acantamoeba infection. Its fungicide activity was recently shown by its lethal effect on yeasts that contaminate the industrial ethanol process, and on the PE-2 strain of Saccharomyces cerevisiae, one of the main fermenting yeasts in Brazil. This pointed to the need to know the molecular mechanism that lay behind the cell resistance to this compound. In this study, we examined the factors involved in PHMB-cell interaction and the mechanisms that respond to the damage caused by this interaction. To achieve this, two research strategies were employed: the expression of some genes by RT-qPCR and the analysis of mutant strains.

Results: Cell Wall integrity (CWI) genes were induced in the PHMB-resistant Saccharomyces cerevisiae strain JP-1, although they are poorly expressed in the PHMB-sensitive Saccharomyces cerevisiae PE2 strain. This suggested that PHMB damages the glucan structure on the yeast cell wall. It was also confirmed by the observed sensitivity of the yeast deletion strains, Δslg1, Δrom2, Δmkk2, Δslt2, Δknr4, Δswi4 and Δswi4, which showed that the protein kinase C (PKC) regulatory mechanism is involved in the response and resistance to PHMB. The sensitivity of the Δhog1 mutant was also observed. Furthermore, the cytotoxicity assay and gene expression analysis showed that the part played by YAP1 and CTT1 genes in cell resistance to PHMB is unrelated to oxidative stress response. Thus, we suggested that Yap1p can play a role in cell wall maintenance by controlling the expression of the CWI genes.

Conclusion: The PHMB treatment of the yeast cells activates the PKC1/Slt2 (CWI) pathway. In addition, it is suggested that HOG1 and YAP1 can play a role in the regulation of CWI genes.

Figure 1

Relative expressions of the yeast genes involved in the cell wall integrity mechanism and in the general stress response. Relative Quantity represents the expression of the studied genes in the PHMB-treated cells (0.01%) over non-treated cells of the two industrial strains JP1 (red column) and PE-2 (blue column).

Figure 2

Cytotoxicity assay of yeast CWI mutants to different doses of PHMB. The percentage of viable cells after treatments refers to the number of CFU in treated samples over compared with non-treated samples of a given strain.

Figure 3

Cytotoxicity assay to PHMB of yeast strains with deletion in oxidative stress response genes. (A) Spot test assay of parental BY4741 and Δyap1 mutants to oxidizing compound H₂O₂ and to PHMB. (B) Dose-response cell survival in a PHMB assay of ctt1, tsa1 and yap1 mutant strains.

Figure 4

Cytotoxicity assay to PHMB of yeast strains with deletion in oxidative stress response genes. (A) Spot test assay in YPD plates in the presence and absence of PHMB. (B) Determination of relative sulfhydryl groups in soluble proteins (PB) and non-proteins (NP) of molecules in the cell extract of BY4741 parental yeast cells and Δyap1 mutant strains in the presence of 0.005% PHMB and 5 mM H₂O₂.

Figure 5

Relative Quantity of yeast genes involved in the cell wall integrity mechanism and the general stress response of parental BY4741 (green column) and Δyap1 (yellow column), strains in response to treatments with 0.005% PHMB (A) and heat shock at 42°C (B).