Specific cell wall proteins confer resistance to nisin upon yeast cells

Abstract

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104-3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.

FIG. 1

S. cerevisiae synchronized by α-factor. Line A, nonbudding cells; line B, cells with small buds; line C, budding cells with migrating nuclei; line D, large budded binuclear cells.

FIG. 2

Correlation of cyclic nisin sensitivity with expression of genes coding for cell wall proteins and proteins involved in yeast cell wall biosynthesis. The levels of expression were determined relative to the level of expression of the ACT1 gene, which did not vary during the cell cycle. (a) Effect of nisin as assessed by determining the percentage of PI-positive cells. The different stages in the cell cycle are indicated at the bottom. (b) Expression of the mRNA of the CWP1 (■) and CWP2 (•) genes and nisin sensitivity (○) through three generations of synchronous growth. (c) Expression of the mRNA of the FKS1 (■) and CHS3 (•) genes and nisin sensitivity (○) through three generations of synchronous growth.

FIG. 3

Nisin sensitivity of wild-type and cell wall protein-deficient yeast strains at an OD₆₂₀ of 1.0. (A through D) Images obtained with the CSLM of membrane integrity and cell viability after treatment with nisin (30 μg/ml). (A) Parent. (B) cwp1Δ. (C) cwp2Δ. (D) cwp1cwp2Δ. (E) Percentages of cells affected by nisin (0 to 50 μg/ml), as inferred from the images shown in panels A through D.

FIG. 3

Nisin sensitivity of wild-type and cell wall protein-deficient yeast strains at an OD₆₂₀ of 1.0. (A through D) Images obtained with the CSLM of membrane integrity and cell viability after treatment with nisin (30 μg/ml). (A) Parent. (B) cwp1Δ. (C) cwp2Δ. (D) cwp1cwp2Δ. (E) Percentages of cells affected by nisin (0 to 50 μg/ml), as inferred from the images shown in panels A through D.

FIG. 4

Nisin sensitivity of pmt1Δ and its parent, SEY6210. The values were deduced from images similar to those shown in Fig. 3A through D.

FIG. 5

Levels of mRNA of the cell wall protein-encoding genes CWP1 and CWP2 in several mutant strains and their corresponding parent strains. All values were normalized to the levels of expression of the ACT1 gene. Subsequently, the level of expression of CWP2 in SU50 was defined as 100%. All other levels of expression were calculated relative to this value.