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Applied and Environmental Microbiology, October 1998, p. 4047-4052, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Specific Cell Wall Proteins Confer Resistance to
Nisin upon Yeast Cells
S. K.
Dielbandhoesing,1
H.
Zhang,1
L. H. P.
Caro,2
J. M.
van
der Vaart,1
F. M.
Klis,2
C. T.
Verrips,1,3 and
S.
Brul1,*
Unilever Research Laboratorium Vlaardingen,
3133 AT Vlaardingen,1
Department of
Molecular Cell Biology, BioCentrum Amsterdam, University of Amsterdam,
1098 SM Amsterdam,2 and
Department of
Molecular Cell Biology, Utrecht University, 3584 CH
Utrecht,3 The Netherlands
Received 30 March 1998/Accepted 11 June 1998
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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.
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INTRODUCTION |
Nisin is an antimicrobial peptide
produced by lactococci and has been used in consumer products for many
years (30). Although this lantibiotic is inhibitory to
microorganisms, it is harmless to humans (15, 16). Nisin is
the first antimicrobial peptide with "generally recognized as safe"
status in the United States for use in processed cheese; in addition,
its use in various food products is allowed in several countries
(9). Nisin is also of interest to the pharmaceutical
industry (10).
Nisin is known to inhibit the growth of a number of gram-positive
bacteria and also the outgrowth of spores of bacilli and clostridia
(15, 16). Furthermore, the gram-negative bacterium Escherichia coli becomes sensitive to nisin when its outer
membrane is made permeable by osmotic shock (20). Inhibition
of the growth of other gram-negative bacteria can be achieved by
simultaneous treatment with nisin and an agent which modifies and
chelates the outer membrane, such as EDTA (29). These
findings are consistent with the notion that nisin acts on the
cytoplasmic membrane. Indeed, the main antimicrobial activity of nisin
seems to rely on the ability of the compound to form pores in the
cytoplasmic membrane, which leads to a loss of small intracellular
molecules and ions and a collapse of the proton motive force (1,
6, 14, 20, 24, 25, 34). To exert its antimicrobial activity,
nisin does not seem to require a specific receptor but instead requires a sufficient trans-negative electrical membrane potential (24, 25). Driessen et al. concluded that nisin acts as an anion
carrier in the absence of anionic phospholipids (12). It has
been suggested that pore formation by nisin in vivo involves local
perturbation of the bilayer structure and
trans-membrane-potential-dependent reorientation from a surface-bound
configuration to a membrane-inserted configuration. In this
membrane-inserted form the hydrophilic side of nisin and the attached
lipid head groups face the center of a water-filled pore. The
hydrophobic surface of nisin and the fatty acid chains of the lipids
point to the lipid bilayer (31).
Nisin has no antimicrobial effect on yeasts and filamentous fungi.
These organisms each have a rigid cell wall, a complex structure
consisting of glucan cross-linked with chitin and cell wall proteins
(4, 18). The processing of mannoproteins is complex and has
been partially characterized in yeasts (19, 21). A similar
mechanism has been suggested for filamentous fungi (4).
Because mannoproteins are generally considered one of the key wall
components which determine cell wall porosity (8, 18), they
may represent a major barrier preventing free permeation of nisin
through the cell wall and thus access to the cytosolic membrane.
Initial experiments indicated that a yeast cell is prone to the
antimicrobial activity of nisin in certain stages of the cell cycle,
suggesting that cell cycle-regulated components of the cell envelope
are involved. Recently, Caro et al. showed that specific cell wall
mannoproteins are expressed in different stages of the cell cycle
(7). In this study we assessed the importance of individual
mannoproteins, 
-1,3-glucan, and chitin in conferring resistance to
nisin upon yeast cells. In addition, below we present data describing
the role of cell wall protein 1 (Cwp1p) and Cwp2p in the structuring of
a normal yeast wall.
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MATERIALS AND METHODS |
Strains, probes, and media.
The E. coli strain
used in this study was JM109 {endA1 recA1 gyrA96 thi
hsdR17 (rK
mK+)
relA1 supE44 (lac-proAB) [F' traD36 proAB
lacqZM15]} (35), which was
grown in Luria broth (26) supplemented with 100 µg of
ampicillin/ml when appropriate. The Saccharomyces cerevisiae
strains used were SU50 (YT6-2-1 L) (MATa
cir0 leu2-3,112 his4-519 can1) and
SU51 (MATa cir+ leu2-3,112 his4-519
can1) (13). Deletion mutants cwp1
cwp2 and cwp1cwp2 derived from SU50 were kindly provided by
J. M. van der Vaart (32). Deletion mutant
cwh53 and its parent, AR27 (MATa
ura3-52), were a kind gift from A. F. J. Ram
(23). Deletion mutant pmt1 and its parent,
SEY6210 (MATa leu2-3,112 ura3-52 his-200 lys2-801
trp1-901 suc-9), were a gift from J. P. Bourdineaud
(3). Yeast strains were maintained on YPD agar (1% yeast
extract, 2% Bacto Peptone, 2% glucose, 1.5% Bacto Agar) or synthetic
minimal medium agar containing 0.7% yeast nitrogen base, 2% glucose,
1.5% Bacto Agar, and amino acids as necessary (27). The
yeast cells used for all assays were precultured on YPD agar.
Plasmids pUR2984-CWP1, pUR2984-CWP2, pUR2984-SED1, and pUR2984-TIP1
were kind gifts from J. M. van der Vaart (32). Plasmids pSB4 containing the FKS1 gene and plasmid pCHS3 containing
the CHS3 gene were kind gifts from A. F. J. Ram
(23) and J. H. Vossen (22). Plasmid pH2A was
kindly provided by H. Sillje (33).
Probe preparation.
Plasmids were digested with
NheI and HindIII. The DNA fragments
containing part of the CWP1 gene (642 bp), part of the
CWP2 gene (204 bp), part of the SED1 gene (690 bp), or part of the TIP1(435 bp) gene were isolated from an
agarose gel. pH2A was cut with SacI, and pCHS3 was cut with
BamHI, which resulted in probes H2A (2.3 kb) and
CHS3 (2.8 kb). The probe fragment of the FKS1
gene (2.1 kb) was isolated from pSB4 as a
ClaI-XhoI fragment. All fragments were purified
by QIAEX 150 gel extraction. The specific DNA probes were randomly
labelled by using [
32-P]dCTP (Amersham) as a substrate
(26).
Reagents.
Nisin was obtained from Aplin & Barret (Dorset,
United Kingdom). Yeast nitrogen base, Bacto Peptone, Bacto Yeast
Extract, and Bacto Agar were obtained from Difco Laboratories (Detroit, Mich.). DNA restriction enzymes were purchased from New England Biolabs
Inc. (Beverly, Mass.) and Boehringer Mannheim Biochemicals (Mannheim,
Germany). -Factor was obtained from Bachem Feinchemikalien AG.
4',6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma Chemical
Co. (St. Louis, Mo.). Propidium iodide (PI), Calcofluor White, and FUN1
were obtained from Molecular Probes Inc., European BV (Leiden, The
Netherlands). The [32-P]dCTP used for DNA probe
labelling and the Hybond-N membrane used for Northern blotting were
obtained from Amersham (Arlington Heights, Ill.). Zymolyase 100T was
obtained from Seikagaku Kogyo Co. (Tokyo, Japan).
Analytical procedures.
The confocal scanning laser
microscope (CSLM) used consisted of a Zeiss Axioplan inverted
microscope, a Bio-Rad model MRC-1024 system, and Lasersharp software.
The objective used was a 1.5× zoom objective (magnification, ×63)
with an image width of 110 µm. The software used to determine the
percentage of PI-positive cells was the Leica Q500 MC software, as
adapted by Aat Don (Department of Analytical & Information Sciences,
Unilever, Vlaardingen, The Netherlands).
Yeast spheroplast generation.
Yeast cells were harvested by
centrifugation and washed twice in 10 mM Tris-HCl (pH 7.4) and once in
spheroplasting buffer (50 mM Na2CO3 [pH 7.4],
1 M sorbitol). The washed cells were resuspended in 10 ml of
spheroplasting buffer, and 20 µl of -mercaptoethanol was added.
After 10 min of incubation at room temperature, 200 µl of Zymolyase
100T (5 mg/ml) was added, and the preparation was incubated at 37°C
for an additional 40 min. The quality of spheroplast formation was
assessed by diluting the preparation in deminerilized water
(dH2O) and determining the percentage of spheroplasts that
lysed.
Yeast culture synchronization.
Yeast cells were grown in YPD
medium for 14 h at 25°C to an optical density at 620 nm
(OD620) of 0.3. Subsequently, the culture was incubated
with -factor at a final concentration of 4 mg/ml at 25°C for
2.5 h. After induction, the cells were washed twice in YPD medium
and grown under the same conditions for further analysis. The time that
cells were transferred to fresh medium was considered zero time.
Samples were taken every 30 min and used to determine the level of
synchronization, the nisin sensitivity (determined by calculating the
percentage of cells that were stained with PI), and the mRNA levels of
several proteins (normalized to the level of ACT1).
The cell cycle stage was assessed by using fluorescence microscopy.
Yeast cells were fixed in 0.13% formaldehyde, centrifuged, and
resuspended in 96% (vol/vol) ethanol. Cell nuclei were visualized by
staining cells in the dark with 1 µg of DAPI per ml for 15 min
(17). Subsequently, after two washes with dH2O,
the four cell-cycle stages were identified by using fluorescence
microscopy.
Nisin sensitivity.
Yeast cells harvested from a synchronous
culture were resuspended to an OD620 of 1.0. Then 100 µl
of the suspension was incubated with 10 µg of nisin per ml at pH 4 at
room temperature for 2 min. In the experiments in which the nisin
sensitivities of cells and protoplasts were compared, a nisin
concentration of 80 µg/ml was used. In the experiments performed with
cell wall mutants, a concentration range of 0 to 50 µg/ml was
assessed. After the peptide was removed, the pellet was suspended in 90 µl of dH2O and incubated with 5 µl of 100 µM FUN1 at
30°C for 20 min and then with 5 µl of a 1-mg/ml PI solution for 10 min under the same conditions (5). After staining, the cells
were washed twice with dH2O to remove the excess dye and
were analyzed with the CSLM system.
RNA isolation and Northern hybridization.
RNA was isolated
by the hot phenol extraction method without the use of glass beads, as
described elsewhere (1a). Basically, cells were lysed in TES
solution (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 0.5% sodium dodecyl
sulfate) and vigorously vortexed after incubation in water-saturated
phenol at 65°C for 1 h. The aqueous phase was extracted once
with phenol and once with chloroform. The RNA was precipitated with
ethanol.
For Northern blotting (
7,
17), 10 µg of RNA was loaded
onto a 1% RNA agarose gel system containing formaldehyde and
formamide.
After blotting, the RNA was cross-linked to Hybond-N+
membranes
by UV radiation. Northern hybridizations were performed in
the
presence of 50% formamide at 42°C by using
32-P-labelled gene fragments. The blots were washed at
42°C with
decreasing concentrations of SSC (1× SSC is 0.15 M NaCl
plus 15
mM sodium citrate, pH 7.0) down to 0.5× SSC in the presence of
0.1% sodium dodecyl sulfate. Hybridization signals were quantified
by
scanning autoradiograms in the linear range of the films. Levels
of
expression were normalized to actin levels.
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RESULTS |
Yeast wall forms a barrier for small antifungal peptides.
In
order to determine whether the yeast cell wall forms a barrier to
nisin, we first incubated cells with EDTA and dithiothreitol as
described by de Nobel et al. (11) in order to increase the wall permeability. We observed that treatment of log-phase yeast cells
with EDTA and/or dithiothreitol made them significantly more sensitive
(by a factor of 2 to 4) to treatment with small antimicrobial peptides,
such as nisin, as measured by the increase in the percentage of
PI-positive cells. To study the inferred barrier function of the yeast
wall for nisin in more detail, we removed the cell wall by incubation
with a wall-lytic enzyme preparation and incubated the spheroplasts
with nisin. Spheroplasts did not stain with Calcofluor White but were
stained when they were incubated with the viability dye FUN1. Cells
with damaged membranes were stained red due to uptake of PI. Yeast
spheroplasts rapidly lysed when they were incubated in the presence of
nisin at concentrations which hardly affected intact cells (10 to 80 µg/ml). Apparently, the cell wall normally forms a barrier for nisin.
As the composition of the cell wall varies during the cell cycle, we
set out to analyze the nisin sensitivity of yeast cells during the
cycle.
Nisin sensitivity during the yeast cell cycle.
After
incubation of a yeast culture with -factor, the synchronous growth
of this culture was checked (Fig. 1).
Three synchronous consecutive cell cycles were observed. Confirmation
of the cell cycle progression was obtained by measuring the fluctuation
in H2A mRNA levels. The level of the mRNA of H2A,
a prominent cell cycle marker gene which is actively transcribed in the
S phase, peaked at 60, 150, and 240 min after the transfer to fresh
medium without -factor. This is consistent with the observation that cells had small buds at these time points.
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FIG. 1.
S. cerevisiae synchronized by -factor.
Line A, nonbudding cells; line B, cells with small buds; line C,
budding cells with migrating nuclei; line D, large budded binuclear
cells.
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The nisin sensitivity of the synchronous culture is shown in Fig.
2a. In the first cycle high percentages
of PI-positive cells
were recorded, and the culture was dominated by
cells with small
buds. In the second and third generations, however,
cells with
migrated nuclei seemed to be most sensitive to nisin.
Perhaps
the cells in the first cycle still suffered from direct effects
of
-factor on the structure of their cell walls.
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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.
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Figure
2b shows the transcription of
CWP1 and
CWP2 in relation to culture sensitivity to nisin. Maximum
transcription of
CWP1 was observed during the second and
third generations, at the stage
in the cell cycle when cells with small
buds dominated the culture
(150 and 240 min). Essentially similar
results were obtained for
SED1 (data not shown). The level
of the mRNA of
CWP2, on the other
hand, started to peak in
the first generation at the G2 phase
of the cycle (90 min). A similar
trend was observed in the second
and third cycles (180 and 270 min). Finally, the level of
TIP1 expression started high,
dropped, and subsequently peaked early
in the G1 phase at cell cycle
stages just before cell division
(data not shown).
Figure
2c shows the pattern of expression of
FKS1, the gene
coding for the catalytic subunit of the
-1,3-glucan-synthesizing
complex, which is expressed when cells are cultured in
glucose-containing
media. Clearly, treatment with
-factor
down-regulated expression
of this gene. After release of the G1 block,
the mRNA levels gradually
increased again. A similar situation occurs
with chitin synthase
3 gene expression. Chs3p is responsible for chitin
synthesis in
the chitin ring and in lateral walls.
The maximal levels of
CWP1 and
SED1 mRNA in the
second and third generations were followed by maximal cell sensitivity
to
nisin. In contrast, peaks in
CWP2 transcription were
followed
by maximal cell resistance to nisin. No clear correlation of
TIP1 expression with nisin sensitivity was observed,
although a tendency
towards a high level of expression being followed
by a high level
of resistance to nisin was noted. Nor was there a
correlation
between the levels of expression of
FKS1 and
CHS3 and the cyclic
sensitivity to nisin. Thus, we concluded
that glucan and chitin
levels as such were not important in the
protection of yeast cells
from nisin, whereas Cwp2p seemed to be very
important in conferring
resistance to nisin upon yeast cells. These
inferred roles were
further substantiated by an analysis of nisin
sensitivity in which
various knockout yeast mutants were used. We
analyzed a
cwp1 mutant yeast strain, a
cwp2
mutant yeast strain, a
cwp1cwp2 mutant yeast strain, a
pmt1 mutant yeast strain, and a
cwh53(fks1) mutant yeast strain.
Sensitivity of yeast cell wall mutants to nisin.
Figure
3 shows that logarithmically grown
cwp1 cells were clearly more sensitive to the peptide
than wild-type yeast cells were. cwp2 cells were even
more sensitive to nisin. Finally, almost all of the cells of the double
mutant were sensitive to nisin, as shown by their massive uptake of PI.
Figure 3E shows the results in a bar diagram for nisin concentrations
up to 50 µg/ml. A similar analysis was subsequently performed with a
pmt1 strain. Bourdineaud et al. have recently shown that
this strain contains exceptionally low amounts of glucanase-extractable
mannoproteins in its cell wall and accordingly has increased wall
permeability, as shown by its hypersensitivity to Zymolyase treatment
(3). However, pmt1 cells were not
hypersensitive to nisin (Fig. 4). As
Cwp2p does occur in the walls of pmt1 cells, although at
low levels, we concluded that Cwp2p plays a more crucial role than other glucanase-extractable mannoproteins in structuring the cell wall
in such a way that the cell is protected against nisin. Fks1p-deficient cells, whose walls have a much lower -1,3-glucan level, did not exhibit increased nisin sensitivity, confirming that glucan layers as
such do not play a major role in the prevention of nisin permeation through the cell wall (data not shown).
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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)
cwp1. (C) cwp2. (D) cwp1cwp2.
(E) Percentages of cells affected by nisin (0 to 50 µg/ml), as
inferred from the images shown in panels A through D.
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FIG. 4.
Nisin sensitivity of pmt1 and its parent,
SEY6210. The values were deduced from images similar to those shown in
Fig. 3A through D.
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Regulation of CWP1 and CWP2 expression in
cell wall mutants.
To investigate the proposed important function
of CWP1 and CWP2 in the biogenesis of a normal
yeast cell wall, we analyzed the levels of CWP1 and
CWP2 transcripts in various yeast cell wall mutants and
their parents. Figure 5 shows the
results. Clearly, in a Cwp1p-deficient strain the levels of expression
of CWP2 were also lower than the levels of expression in the
wild-type strain. In contrast, the transcription of CWP1 in
a Cwp2p-deficient strain increased. This suggests that in the analysis
of the antifungal effect of nisin on cwp1 described
above, the effect of nisin on the mutant due to the absence of Cwp1p
could be overestimated since the level of Cwp2p was also decreased. On
the other hand, it was evident in the analogous analysis of
cwp2 that the effect of nisin on this mutant could not be
ascribed to a decrease in CWP1 expression.
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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.
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Interestingly, in the Pmt1p-deficient strain expression of
CWP2 was induced significantly, whereas in the
Fks1p-deficient
strain expression of both
CWP1 and
CWP2 was induced moderately.
We found that in the
fks1 mutant, in addition to both cell wall
protein genes,
chitin synthase expression was also induced (data
not shown). In this
mutant the cell wall proteins are anchored
mainly in the cell wall to
the extra chitin (
17c). Neither
SED1 nor
TIP1 was induced in these strains. Together, the data
strongly
suggest that Cwp2p and Cwp1p have an important structural
function
in the yeast cell wall (
17a).
| |
DISCUSSION |
Our studies show that specific glucanase-extactable cell wall
proteins with no known physiological function are crucial in conferring
resistance to the antimicrobial peptide nisin upon yeast cells. As the
yeast cell wall mannoproteins are heavily glycosylated and therefore
determining their specific levels immunologically is very difficult, we
chose to measure the level of transcription of the genes involved in
the expression of cell wall proteins rather than work with the proteins
themselves. Although transcription levels do not necessarily correlate
with the presence of the components in the cell wall, we know from
preliminary studies performed with green fluorescent protein fusions
that Cwp1p and Cwp2p appear in the cell wall at distinct points in the
cell cycle, in agreement with Northern analysis data (17b).
High levels of transcription of Cwp2p just before the stage in the cell
cycle when the cells were very resistant to nisin suggested that this
protein protects the cell from nisin and similar peptides. Upon
depletion of both Cwp2p and Cwp1p, the cells were very sensitive to
nisin, as demonstrated by the high percentage of PI-positive cells. The
fact that expression of CWP1 was induced in a
cwp2 strain whereas CWP2 expression was not
induced in a cwp1 strain and the fact that
cwp2 cells were slightly more sensitive to nisin than
cwp1 cells support the conclusion that the Cwp2p protein
seems to be more important than the Cwp1p in conferring nisin
resistance upon yeast cells. van der Vaart et al. (32)
showed that the exponentially growing cwp2 deletion mutant
is more sensitive to Calcofluor White, Congo red, and Zymolyase than
the cwp1, tip1, and srp1 deletion
mutants. Furthermore, depletion of Cwp2p resulted in a thinner
electron-dense layer around the glucan layer. The importance of Cwp2p
for normal cellular physiology is also underlined by the observation
that overexpression of this protein can partially compensate for the lack of sphingolipids. Sphingolipids are necessary for the growth of
S. cerevisiae bypass mutants at low pH values
(28). Survival of these mutants at low pH values is enhanced
by overexpression of the Cwp2 protein.
In addition to nisin, there are similar small, membrane-perturbing
peptides, such as histatins, cecropins, and magainins, as well as
synthetically modelled peptides (2). We studied the
antifungal activity of synthetically produced peptides during the yeast
cell cycle with the approach described in this paper and found a
pattern similar to that observed for nisin (11a). Yeast cell
wall proteins are also involved in resistance to somewhat larger
membrane-active plant antimicrobial proteins. Yun et al. (36) recently showed that Pir cell wall proteins are induced in yeast cells in response to a challenge with the plant antifungal PR-5 protein osmotin.
We are currently constructing fusion proteins which consist of
-galactosidase, a protease-processing site, and various Cwps. These
constructs should allow us to purify Cwps with (parts of) their
glycosyl phosphatidyl inositol anchors for peptide binding studies.
Furthermore, we plan to perform photolabelling experiments with nisin
(and other peptides) in incubations with yeast cells. Complexes formed
upon cross-linking of the peptides to the yeast wall will be analyzed
in order to characterize the in vivo binding of the peptides to wall
components. These two approaches should allow us to distinguish between
a direct protective effect of Cwps against peptides through specific
binding and indirect protection through an influence on the structural
organization of the wall.
Finally, how yeast cells respond to a constant challenge with an
antimicrobial peptide is not yet clear. Another issue is whether the
age of yeast cells influences their sensitivity to peptides; this could
explain the apparent sensitivity of some wild-type cells to nisin, as
indicated by the data in Fig. 3A. Current studies are aimed at
answering these questions.
| |
ACKNOWLEDGMENT |
John Chapman is gratefully acknowledged for his help during this
study and for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Exploratory
Research Foods Group, Unilever Research Laboratory Vlaardingen, Olivier van Noortlaan 120, Vlaardingen, South-Holland 3133 AT, The Netherlands. Phone: 31-10-4605161. Fax: 31-10-4605188. E-mail:
stanley.brul{at}unilever.com.
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Abstract
Introduction
