Previous Article | Next Article 
Applied and Environmental Microbiology, December 1999, p. 5451-5458, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Permeabilization of Fungal Membranes by Plant
Defensins Inhibits Fungal Growth
Karin
Thevissen,
Franky
R. G.
Terras,
and
Willem F.
Broekaert*
F. A. Janssens Laboratory of Genetics,
Katholieke Universiteit Leuven, B-3001 Heverlee-Leuven, Belgium
Received 4 March 1999/Accepted 15 September 1999
 |
ABSTRACT |
We used an assay based on the uptake of SYTOX Green, an organic
compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced
by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 µM), strong permeabilization was detected that could be strongly
suppressed by cations in the medium. This permeabilization appears to
rely on direct peptide-phospholipid interactions. At lower defensin
levels (0.1 to 1 µM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the
inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the
plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia
merckii, induced cation-resistant membrane permeabilization
in yeast (Saccharomyces cerevisiae) which correlated with
its antifungal activity. However, Dm-AMP1 could not induce
cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically
reduced capacity for binding Dm-AMP1. We think that
cation-resistant permeabilization is binding site mediated and
linked to the primary cause of fungal growth inhibition induced by
plant defensins.
| |
INTRODUCTION |
Plant defensins are a family of
small (45 to 54 amino acids), usually basic, peptides occurring in
various plant species (2, 3). Many plant defensins can
inhibit the growth of a broad range of fungi at micromolar
concentrations but are nontoxic to both mammalian and plant cells
(18, 19). In some plant tissues, the expression of defensin
genes is induced in response to fungal infection (21),
whereas in other tissues they are expressed constitutively
(31). All plant defensins share a common three-dimensional folding pattern, stabilized by eight disulphide-linked cysteines (2, 3). Plant defensins are structurally related to
antibacterial insect defensins (8) and drosomycin, an
antifungal peptide found in insects (15). The plant defensin
family may be divided into two main groups (A and B), sharing only 25%
similarity (11). Group A can be further divided into four
subfamilies (A1, A2, A3, and A4) with at least 50% similarity within
each subfamily. Members of subfamily A2, formerly termed nonmorphogenic
plant defensins (3), including Dm-AMP1 from Dahlia
merckii, reduce hyphal elongation without affecting fungal
morphology. In contrast, members of subfamilies A3 and A4, including
Rs-AFP2 from Raphanus sativus and Hs-AFP1 from
Heuchera sanguinea, respectively, induce tip ballooning and
branch formation on susceptible fungi (19). These plant
defensins have therefore been termed morphogenic plant defensins
(3).
Plant defensins induce ion fluxes across the plasma membranes of living
fungal hyphae (28). Unlike insect (8) and
mammalian (12) defensins, plant defensins neither form
ion-permeable pores in artificial membranes nor change the electrical
properties of artificial lipid bilayers (28). This finding
indicates that a direct interaction with lipid components of the plasma
membrane, a mechanism proposed to explain the antimicrobial effects
of insect defensins or mammalian defensins (8, 12), is
unlikely for plant defensins. In addition, specific, high-affinity
binding sites for plant defensins on fungal cells and microsomal
membranes have been identified based on studies with radiolabeled plant defensins (29, 30). Binding of plant defensins to fungal
cells and plasma membrane fractions is partially reversible. A mutant of the yeast Saccharomyces cerevisiae has been identified
which, in contrast to the wild-type strain, is not sensitive to the
plant defensin Dm-AMP1. The capacity of this mutant, called DM1, to bind Dm-AMP1 to its plasma membrane is more than 10-fold less than that
of the wild type, suggesting that binding of Dm-AMP1 to a specific
binding site is a prerequisite to the antifungal activity of this plant
defensin (30). Combined, these observations suggest that the
ion fluxes may result from (i) the interaction of the plant defensins
with a binding site that transduces a signal to endogenous ion channels
in the membrane or (ii) binding-site-mediated insertion of the plant
defensins into the membrane, with subsequent ion channel formation.
To distinguish these models, we studied the influx of
SYTOX Green, a high-affinity nucleic acid stain that
fluoresces upon nucleic acid binding and for which no endogenous
channels exist. SYTOX Green penetrates cells with compromised
plasma membranes but does not cross the membranes of noncompromised
living cells (17, 23, 34). SYTOX Green uptake was
measured fluorimetrically in Neurospora crassa and
S. cerevisiae treated with different plant
defensins. Our results are consistent with a model for plant defensin
action involving binding-site-mediated insertion of the defensins into
the plasma membrane.
| |
MATERIALS AND METHODS |
Materials.
The antifungal peptides Dm-AMP1, Hs-AFP1, and
Ah-AMP1 were isolated as described previously (19). Rs-AFP2,
Rs-AFP2(Y38G), and the membrane-active antifungal 
-purothionin
(-PT) found in wheat seeds were isolated as described previously by
Terras et al. (26), De Samblanx et al. (9), and
Redman and Fisher (22), respectively. SYTOX Green was
obtained from Molecular Probes (Eugene, Oreg.). Carbonyl cyanide
m-chlorophenylhydrazone (CCCP) was purchased from Sigma (St.
Louis, Mo.). All other reagents were of reagent grade and were obtained
from commercial sources.
Microorganisms.
N. crassa FGSC 2489 was grown on
six-cereal agar, and conidia were harvested as described previously
(4). Conidium stocks in 20% (vol/vol) glycerol were at a
final concentration of 2 × 107 conidia/ml. Yeast
(S. cerevisiae) was grown and stored following standard
protocols (1). S. cerevisiae strains used were
W303-1A (genotype, MATa leu2-3/112 ura3-1 trp1-1
his3-11/15 ade2-1 can1-100 GAL SUC2) and the W303-1A-derived
mutant DM1 (30).
Antifungal activity assay.
Activity against N. crassa was assayed by microspectrophotometry (4, 26).
N. crassa was grown at an inoculum density of 3 × 105 conidia/ml in 12 g of potato dextrose broth per
liter (Difco, Detroit, Mich.) with continuous shaking (200 rpm) at
22°C. After 20 h of incubation, hyphae were washed with
either SMF1 or SMF1 supplemented with either 50 mM KCl or 5 mM MgCl2 · 6H2O, (SMF1 is synthetic medium
for fungi, containing 50 µM MgSO4 · 7H2O, 50 µM CaCl2 · 2H2O, 5 µM
FeSO4 · 7H2O, 0.1 µM CoCl2,
0.1 µM CuSO4 · 5H2O, 2 µM
Na2MoO4 · 2H2O, 0.5 µM
H3BO3, 0.1 µM KI, 0.5 µM
ZnSO4 · 7H2O, 0.1 µM
MnSO4 · 1H2O, 10 g of glucose per
liter, 1 g of asparagine per liter, 20 mg of methionine per liter,
2 mg of myo-inositol per liter, 0.2 mg of biotin per liter,
1 mg of thiamine-HCl per liter, 0.2 mg of pyridoxine-HCl per liter, 0.5 mM K2HPO4 · 3H2O). Ninety-microliter aliquots of the suspension of N. crassa
hyphae in the appropriate medium were mixed with 10 µl of the same
medium containing antifungal peptides and incubated in transparent
96-well microtiter plates. After incubation for 24 h at 22°C
without shaking, the absorbance at 595 nm was determined with an
enzyme-linked immunosorbent assay reader (26). The
absorbance values served as a measure of microbial growth
(26). The concentration of the antifungal protein that is
required to inhibit 50% of the fungal growth was calculated from the
dose-response curves with twofold dilution steps (26).
The activity of protein samples against S. cerevisiae was
determined in an analogous manner. Briefly, 10 µl of the protein sample was mixed in a well of a 96-well microplate with 90 µl of
yeast minimal medium (YMM) (0.8 g of complete supplement mixture [CSM] per liter [BIO 101, La Jolla, Calif.], 6.5 g of yeast
nitrogen base without amino acids per liter [Difco], 20 g of
glucose per liter), containing about 2 × 106 yeast
cells per ml. The microplates were incubated at 30°C without shaking,
and the absorbance at 595 nm was recorded after 20 h of incubation.
SYTOX Green uptake assay.
N. crassa was grown in
potato dextrose broth as described above. After 20 h of
incubation, hyphae were washed with either water buffered with 100 µM
HEPES-KOH (pH 6.5), SMF1, or SMF1 supplemented with either 50 mM KCl or
5 mM MgCl2. Ninety-microliter aliquots of the suspension of
N. crassa hyphae, supplemented with 0.2 µM SYTOX
Green, were mixed with 10 µl of the same medium containing antifungal
peptides and incubated in white 96-well microplates (PE white;
Perkin-Elmer, Norwalk, Conn.). After incubation for 10 min to 6 h
at 22°C with periodic agitation, fluorescence emitted by the cells in
the microplates was measured with a Perkin-Elmer LS 50 B fluorescence
spectrometer at an excitation wavelength of 488 nm (slit, 10 nm) and an
emission wavelength of 540 nm (slit, 5 nm). Fluorescence values of the
samples were corrected by subtracting the fluorescence value of a
culture in the same medium without peptides but with SYTOX Green.
Absolute values of fluorescence did not differ more than 50% in
independent tests performed under identical conditions. SYTOX Green
uptake in S. cerevisiae was measured similarly except that
the medium used was either YMM or YMM supplemented with 5 mM
MgCl2, the cell density was approximately 2 × 108 cells per ml, and incubation was at 30°C.
Fluorescence microscopy.
N. crassa hyphae, grown as
described for the antifungal activity assay, were suspended in SMF1
containing 5 mM MgCl2 and 0.2 µM SYTOX Green, in
either the presence or absence of antifungal peptides. After 360 min of
incubation, fluorescence was viewed with a Nikon Optiphot (Tokyo,
Japan) fluorescence microscope equipped with a B-2A filter set (Nikon)
for fluorescein detection (excitation wavelength, 450 to 490 nm;
emission wavelength, 520 nm).
Statistical analysis.
To determine a possible correlation
between SYTOX Green uptake and antifungal activity, P
values of the corresponding data were calculated with Microsoft Excel
software, by calculating the analysis of variance. If the P
value of the data was lower than 0.05, it was concluded that the data
were significantly correlated.
| |
RESULTS |
Permeabilization of N. crassa and S. cerevisiae suspended in water.
We tested the ability
of the plant defensins to permeabilize N. crassa hyphae in
water (buffered with 100 µM HEPES [pH 6.5]). SYTOX Green
permeabilization was assessed 30 min after the addition of SYTOX
Green and the peptides. -PT, Rs-AFP2, Hs-AFP1, and Dm-AMP1 caused an
increase in SYTOX Green influx (Fig.
1A). At high peptide concentrations (>5
µM), the relative increase in SYTOX Green uptake decreased,
with the greatest decreases for -PT and Rs-AFP2.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 1.
Membrane permeabilization induced by plant defensins and
-PT in N. crassa and S. cerevisiae in water.
Dose-response curves of membrane permeabilization measured by SYTOX
Green fluorescence of N. crassa hyphae (A) and S. cerevisiae wild-type cells (B) are shown. Fungal cells were
suspended in distilled water buffered with 100 µM HEPES (pH 6.5) and
treated with -PT ( ), Rs-AFP2 ( ), Hs-AFP1 (X), or
Dm-AMP1 ( ) for 30 min, whereupon fluorescence was measured. The
dotted line indicates the upper limit of fluorescence detection. Values
correspond to one representative experiment of three.
|
|
Similar observations were made for
S. cerevisiae cells
suspended in water buffered with 100 µM HEPES (pH 6.5).
-PT,
Rs-AFP2,
Hs-AFP1, and Dm-AMP1 all increased the influx of SYTOX
Green (Fig.
1B), with a drop at high concentrations of the plant
defensins.
Dm-AMP1 permeabilized the
S. cerevisiae mutant
DM1, known to be
resistant to Dm-AMP1 and to lack Dm-AMP1 binding sites
(
30),
to essentially the same extent as wild-type cells,
indicating
that this type of permeabilization is not linked to the
presence
of specific binding sites for Dm-AMP1 (Fig.
2).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2.
Membrane permeabilization induced by Dm-AMP1 in S. cerevisiae wild-type cells and S. cerevisiae DM1 cells
in water. Dose-response curves of membrane permeabilization measured by
SYTOX Green fluorescence of S. cerevisiae
wild-type cells ( ) and S. cerevisiae DM1 cells () are
shown. Fungal cells were suspended in distilled water buffered with 100 µM HEPES (pH 6.5) and treated with Dm-AMP1 for 30 min, whereupon
fluorescence was measured. The dotted line indicates the upper limit of
fluorescence detection. Values are averages with standard errors of
triplicate measurements and correspond to one representative experiment
of three.
|
|
Permeabilization of N. crassa in growth medium.
When N. crassa hyphae were suspended in the growth medium
SMF1, membrane permeabilization to SYTOX Green induced by -PT
(Fig. 3A) could be detected after 10 min
of incubation and increased with longer incubation times. A significant
correlation ( = 0.05) between the induced hyphal
permeabilization and the antifungal activity of -PT could be
observed. Weak permeabilization by the plant defensins (Rs-AFP2,
Hs-AFP1, and Dm-AMP1) in the 0.1 to 1 µM range could be detected only
after 2 to 4 h of incubation, whereas stronger permeabilization,
induced at levels greater than 10 µM, could be detected after only 30 min of incubation. The concentrations of plant defensins inducing the
weak permeabilization effect were significantly correlated ( = 0.05) with the concentrations required for growth inhibition.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 3.
Membrane permeabilization and growth inhibition induced
by plant defensins and -PT in N. crassa in low- and
high-ionic-strength growth media. Dose-response curves of membrane
permeabilization measured by SYTOX Green fluorescence and growth
inhibition of N. crassa suspended in SMF1 (A), SMF1 plus 50 mM KCl (B), and SMF1 plus 5 mM MgCl2 (C) are shown. Fungal
hyphae were treated with -PT, Rs-AFP2, Hs-AFP1, or Dm-AMP1, and
fluorescence was measured at different time points. Time points (in
minutes) are indicated at the left of the -PT permeabilization
curves. Values correspond to one representative experiment of three.
|
|
Similar experiments were performed in growth medium with increased
ionic strength, SMF1 supplemented with 50 mM KCl or 5 mM
MgCl
2 (Fig.
3B and C). Previous work has established that
the
presence of monovalent cations and especially of divalent cations
in the growth medium reduces the antifungal activity of plant
defensins, and that little or no antifungal activity is observed
at concentrations of monovalent or divalent cations above 100
or 10 mM,
respectively (
19,
26,
27).
-PT permeabilized
N. crassa irrespective of the ionic strength of the medium, although
the effect was less pronounced in the 5 mM MgCl
2 treatment
than
in the 50 mM KCl treatment. In all tested media, the
dose-response
curves of permeabilization by
-PT correlated
significantly (
= 0.05) with those of growth inhibition,
supporting the earlier
hypothesis that thionins affect fungal
growth by causing membrane
permeabilization (
5,
6,
28).
For the three plant defensins tested, permeabilization depended
upon the cation composition of the medium. The strong permeabilization,
observed after 30 min under low-ionic-strength conditions at plant
defensin concentrations above 10 µM, was drastically reduced in
the presence of either 50 mM KCl or 5 mM MgCl
2. The weak
permeabilization,
detected at lower doses of plant defensins (0.1 to 1 µM), was
not affected or only weakly affected by the presence of
cations
in the medium. In addition, this weaker and more
cation-resistant
permeabilization correlated significantly (
= 0.05) with the
antifungal activity of the plant
defensins.
When viewed with a fluorescence microscope,
N. crassa cells
suspended in SMF1 supplemented with 5 mM MgCl
2 in the
presence
of 20 µM plant defensins showed strong SYTOX Green
fluorescence
in the cytosol and especially in the nuclei (Fig.
4). This observation
confirmed that the
plant defensins cause intracellular uptake
of the dye under these
conditions.
View larger version (67K):
[in this window]
[in a new window]
|
FIG. 4.
Fluorescence microscopy of fungal cells in the presence
of SYTOX Green and Dm-AMP1. N. crassa cells were
suspended in SMF1 supplemented with 5 mM MgCl2 and 0.2 µM SYTOX Green and treated for 360 min in the absence (A) or
presence (B) of 20 µM Dm-AMP1. Upper panels are light microscopic
images; lower panels are fluorescence microscopic images. Bar, 25 µm.
|
|
Carbendazim, an inhibitor of microtubule formation, did not increase
SYTOX Green uptake in the concentration range tested
(0.1 to 1 mM),
although it completely inhibited growth of
N. crassa (results not shown). Hence, growth inhibition by itself is not
sufficient to increase permeabilization of fungal cells to SYTOX
Green.
Permeabilization of various antimicrobial agents has previously
been shown to be abolished by treating cells with a
membrane-depolarizing
agent (
10). Treating
N. crassa cells with plant defensins in
the presence of 2 µM
CCCP significantly reduced permeabilization
to SYTOX Green,
especially with lower doses of plant defensins
(0.1 to 1 µM) (Fig.
5).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 5.
Effect of CCCP on membrane permeabilization induced by
Rs-AFP2 in N. crassa. Dose-response curves of membrane
permeabilization measured by SYTOX Green fluorescence of N. crassa suspended in SMF1 in the absence () or presence ( ) of
2 µM CCCP are shown. Fluorescence was measured 4 h after the
addition of SYTOX Green, CCCP, and Rs-AFP2. Values correspond to
one representative experiment of two.
|
|
Rs-AFP2(Y38G) is a variant of Rs-AFP2 with substantially decreased
antifungal activity (
9). Membrane permeabilization induced
by Rs-AFP2(Y38G) under low-ionic-strength conditions could be
detected
only at concentrations above 10 µM (Fig.
6A). Under high-ionic-strength
conditions, however, no permeabilization could be detected (Fig.
6B),
indicating that Rs-AFP2(Y38G) induces only cation-sensitive
membrane
permeabilization. This cation-sensitive permeabilization
correlates significantly (
= 0.05) with the antifungal activity
of Rs-AFP2(Y38G) in low-ionic-strength medium.
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 6.
Membrane permeabilization and growth inhibition induced
by Rs-AFP2 and Rs-AFP2(Y38G) in N. crassa in low- and
high-ionic-strength growth media. Dose-response curves of membrane
permeabilization measured by SYTOX Green fluorescence and growth
inhibition of N. crassa suspended in SMF1 (A) and SMF1 plus
5 mM MgCl2 (B) are shown. Fungal hyphae were incubated with
Rs-AFP2 () and Rs-AFP2(Y38G) () for 4 h, whereupon
fluorescence was measured. The dotted line indicates the upper limit of
fluorescence detection. Values correspond to one representative
experiment of two.
|
|
Permeabilization of S. cerevisiae suspended in growth
medium.
Rs-AFP2 and Hs-AFP1 did not inhibit growth of
S. cerevisiae in YMM and also failed to cause
permeabilization to SYTOX Green (Fig.
7). Dm-AMP1 induced membrane
permeabilization in S. cerevisiae after 12 h of
incubation, irrespective of the presence of cations in the medium. This
cation-resistant permeabilization correlated significantly ( = 0.05) with the antifungal activity. No such permeabilization was
induced by Dm-AMP1 in the DM1 mutant.
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 7.
Membrane permeabilization induced by plant defensins and
-PT in S. cerevisiae wild-type cells and S. cerevisiae DM1 cells in low- and high-ionic-strength growth media.
Dose-response curves of membrane permeabilization measured by SYTOX
Green fluorescence and growth inhibition of S. cerevisiae
wild-type cells and S. cerevisiae DM1 cells suspended in YMM
(A) and YMM plus 5 mM MgCl2 (B) are shown. Fungal cells
were incubated with Rs-AFP2, Hs-AFP1, or Dm-AMP1 for 12 h,
whereupon fluorescence was measured. Values correspond to one
representative experiment of two.
|
|
| |
DISCUSSION |
Plant defensins have a biphasic effect on the membrane
permeabilization of N. crassa depending on the plant
defensin dose. At high doses, strong permeabilization can be detected
within 30 to 60 min of the addition of the plant defensins. This effect depends on the ionic constitution of the medium: it is most pronounced in water but is almost undetectable in the presence of either 50 mM
K+ or 5 mM Mg2+. In SMF1,
cation-sensitive permeabilization occurs at plant defensin doses approximately 10-fold higher than those causing fungal growth inhibition. These findings are consistent with previous observations that plant defensins cause detectable permeabilization of N. crassa in a low-ionic-strength growth medium to the
nonmetabolizable compound isoaminobutyric acid only at doses that are
an order of magnitude above those required for antifungal activity
(28). Hence, cation-sensitive permeabilization does not
appear to be the primary cause of growth inhibition in hyphae treated
with plant defensins.
At lower plant defensin doses, permeabilization is relatively
weak (as it requires at least 2 to 4 h of incubation to be
detected) and much less affected by either 50 mM K+ or 5 mM
Mg2+. The doses at which weak cation-resistant
permeabilization occurs correlate significantly with those for growth
inhibition, suggesting that this effect is linked to the primary cause
of defensin-induced growth inhibition.
In the case of S. cerevisiae, two types of membrane
permeabilization were observed: (i) a cation-sensitive membrane
permeabilization which is induced by all plant defensins in
water-suspended cells and (ii) a cation-resistant membrane
permeabilization that was observed only with Dm-AMP1. Since the
growth of S. cerevisiae is inhibited only by Dm-AMP1 and not
by Hs-AFP1 or Rs-AFP2 (30), the cation-sensitive
permeabilization cannot play a role in the growth inhibition of
S. cerevisiae cells suspended in growth medium. The doses
required for the cation-resistant membrane permeabilization of S. cerevisiae by Dm-AMP1 in growth medium correlated significantly with those required for antifungal activity, again suggesting that this
kind of membrane permeabilization is linked to the primary cause of
fungal growth inhibition. Although our data clearly reveal similarities
in the way plant defensins act on the yeast S. cerevisiae and the filamentous fungus N. crassa, we do not yet know if
the underlying mechanistic details are the same in both organisms.
Evidence that the first step to fungal growth inhibition by plant
defensins is the binding to a binding site located in the plasma
membranes of fungal hyphae has been previously presented (29,
30). We now propose that the interaction of plant defensins with
such a binding site subsequently enables them to insert into the plasma
membrane, causing a structural disruption and alteration of the
membrane's permeability to ions such as Ca2+ and
K+ and organic molecules like SYTOX Green. This process
of binding-site-mediated membrane insertion and disruption does not
appear to be highly influenced by the presence of cations in the
medium. Binding-site-mediated insertion in plasma membranes has
previously been proposed to explain the antimicrobial activity of a
number of proteins (7, 14, 24, 32). However, this idea has
so far not gained wide support, mainly due to poor experimental evidence.
Our most compelling evidence for binding-site-mediated
cation-resistant membrane permeabilization is the observation that cation-resistant permeabilization was caused by Dm-AMP1 in
wild-type S. cerevisiae but not in the isogenic
Dm-AMP1-resistant DM1 mutant (30). Moreover,
Rs-AFP2(Y38G), a variant of Rs-AFP2 with substantially decreased
antifungal activity, did not induce cation-resistant membrane
permeabilization in N. crassa. Rs-AFP2(Y38G) does not compete with binding sites for Hs-AFP1 on either N. crassa
cells or N. crassa microsomes (29). A single
amino acid substitution might change the three-dimensional
structure of Rs-AFP2 such that interaction with its receptor no
longer occurs. Although the presence of binding sites appears to be a
prerequisite for the antifungal effect of plant defensins under
high-ionic-strength conditions, it does not appear to be sufficient;
S. cerevisiae possesses specific binding sites for
Hs-AFP1, but its growth is not affected by this plant defensin
(20).
Binding of Hs-AFP1 and Dm-AMP1 to fungal microsomes is only partially
reversible (29) and not reversible (30),
respectively. These findings are fully consistent with our model, as
insertion of plant defensins into the plasma membrane following binding would make them inaccessible for competition. The binding of plant defensins to their binding sites is affected by membrane-depolarizing agents (29). Permeabilization to Ca2+
(28) and SYTOX Green (Fig. 5), however, appears to
require a polarized membrane, as these processes are abolished by
treating cells with the membrane-depolarizing agent CCCP. The apparent dependency of permeabilization on membrane polarization might explain
why the dose-response curves for SYTOX Green uptake declined at
doses above those providing maximum effect. This phenomenon was also
observed when fungal cells were treated with detergents such as
sodium dodecyl sulphate or Triton X-100 (results not shown). The
permeabilization of fungal membranes treated with plant defensins will
result in depolarization (28), which may counteract membrane permeabilization. The net result may be strongly influenced by the
plant defensin dose.
At high concentrations of plant defensins (10 to 40 µM), a different
type of permeabilization is observed which is highly suppressible by
cations in the medium. We propose that permeabilization at high plant
defensin doses and low concentrations of cations is due to interaction
with the membrane in a binding-site-independent way. In this case, the
interaction involves only plant defensins and the phospholipid
components of the fungal plasma membranes. Cations, especially divalent
cations, stabilize membrane phospholipid structures (33) and
their presence will counteract the insertion of plant defensins in
membranes. The binding-site-independent nature of this permeabilization
is supported by the observation that permeabilization of
water-suspended S. cerevisiae cells by Dm-AMP1 is equally
strong in the wild type and in the Dm-AMP1-resistant isogenic mutant.
Direct interaction with membrane lipids and subsequent membrane
permeabilization may explain the antimicrobial effects of the
structurally related insect (8) and mammalian
(12) defensins and several linear antimicrobial peptides,
such as magainins, found in Xenopus skin (16);
indolicidin found in the cytoplasmic granules of bovine neutrophils
(13); and sticholysin I, a cytolysin found in the sea
anemone Stichodactyla helianthus (25).
The specificity of plant defensins is dependent on the ionic strength
of the growth medium (19, 26, 27). In
low-ionic-strength medium, plant defensins inhibit most fungi.
This observation is consistent with the proposed
binding-site-independent membrane insertion of plant defensins.
However, in high-ionic-strength medium, the spectrum of plant defensin
activity is narrowed considerably and shows more variation among the
different types of plant defensins. Under these conditions,
binding-site-mediated insertion prevails and the specificity of the
interaction of plant defensins with their binding site dictates the
specificity of the antifungal spectrum of activity.
| |
ACKNOWLEDGMENTS |
This research was supported in part by the Commission of the
European Union (AIR2-CT94-1356) and by a grant from the Fonds voor
Wetenschappelijk Onderzoek
Vlaanderen. K.T. is Postdoctoral Researcher
of the Onderzoeksfonds of the Katholieke Universiteit Leuven, Belgium.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, K. Mercierlaan 92, B-3001 Heverlee-Leuven, Belgium. Phone: 32-16-321631. Fax: 32-16-321966. E-mail:
willem.broekaert{at}agr.kuleuven.ac.be.
Present address: CropDesign, B-9052 Ghent, Belgium.
| |
REFERENCES |
| 1.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman, and K. Struhl.
1993.
Saccharomyces cerevisiae, p. 13.1.1-13.11.4.
In
F. M. Ausubel (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., Somerset, N.Y
|
| 2.
|
Broekaert, W. F.,
B. P. A. Cammue,
M. F. C. De Bolle,
K. Thevissen,
G. W. De Samblanx, and R. W. Osborn.
1997.
Antimicrobial peptides from plants.
Crit. Rev. Plant Sci.
16:297-323.
|
| 3.
|
Broekaert, W. F.,
F. R. G. Terras,
B. P. A. Cammue, and R. W. Osborn.
1995.
Plant defensins: novel antimicrobial peptides as components of the host defense system.
Plant Physiol.
108:1353-1358[Medline].
|
| 4.
|
Broekaert, W. F.,
F. R. G. Terras,
B. P. A. Cammue, and J. Vanderleyden.
1990.
An automated quantitative assay for fungal growth.
FEMS Microbiol. Lett.
69:55-60.
|
| 5.
|
Caaveiro, J. M.,
A. Molina,
J. M. Gonzalez-Manas,
P. Rodriguez-Palenzuela,
F. Garcia-Olmedo, and F. M. Goni.
1997.
Differential effects of five types of antipathogenic plant peptides on model membranes.
FEBS Lett.
410:338-342[Medline].
|
| 6.
|
Carrasco, L.,
D. Vazquez,
C. Hernandez-Lucas,
P. Carbonero, and F. Garcia-Olmedo.
1981.
Thionins: plant peptides that modify membrane permeability in cultured mammalian cells.
Eur. J. Biochem.
116:185-189[Medline].
|
| 7.
|
Chikindas, M. L.,
M. J. Garcia-Garcera,
A. J. Driessen,
A. M. Ledeboer,
J. Nissen-Meyer,
I. F. Nes,
T. Abee,
W. N. Konings, and G. Venema.
1993.
Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.
Appl. Environ. Microbiol.
59:3577-3584[Abstract/Free Full Text].
|
| 8.
|
Cociancich, S.,
A. Ghazi,
C. Hetru,
J. A. Hoffmann, and L. Letellier.
1993.
Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus.
J. Biol. Chem.
268:19239-19245[Abstract/Free Full Text].
|
| 9.
|
De Samblanx, G. W.,
I. J. Goderis,
K. Thevissen,
R. Raemaekers,
F. Fant,
F. Borremans,
D. P. Acland,
R. W. Osborn,
S. Patel, and W. F. Broekaert.
1997.
Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity.
J. Biol. Chem.
272:1171-1179[Abstract/Free Full Text].
|
| 10.
|
Falla, T. J.,
D. N. Karunaratne, and R. E. W. Hancock.
1996.
Mode of action of the antimicrobial peptide indolicidin.
J. Biol. Chem.
271:19298-19303[Abstract/Free Full Text].
|
| 11.
|
Harrison, S. J.,
J. P. Marcus,
K. C. Goulter,
J. L. Green,
D. J. Maclean, and J. M. Manners.
1997.
An antimicrobial peptide from the Australian native Hardenbergia violacea provides the first functionally characterised member of a subfamily of plant defensins.
Aust. J. Plant Physiol.
24:571-578.
|
| 12.
|
Kagan, B. L.,
M. E. Selsted,
T. Ganz, and R. I. Lehrer.
1990.
Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes.
Proc. Natl. Acad. Sci. USA
87:210-214[Abstract/Free Full Text].
|
| 13.
|
Ladokhin, A. S.,
M. E. Selsted, and S. H. White.
1997.
Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids.
Biophys. J.
72:794-805[Medline].
|
| 14.
|
Lakey, J. H.,
F. G. van der Goot, and F. Pattus.
1994.
All in the family: the toxic activity of pore-forming colicins.
Toxicology
87:85-108[Medline].
|
| 15.
|
Landon, C.,
P. Sodano,
C. Hetru,
J. Hoffmann, and M. Ptak.
1997.
Solution structure of drosomycin, the first inducible antifungal protein from insects.
Protein Sci.
6:1878-1884[Medline].
|
| 16.
|
Matsuzaki, K.,
K. Sugishita,
M. Harada,
N. Fujii, and K. Miyajima.
1997.
Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria.
Biochim. Biophys. Acta
1327:119-130[Medline].
|
| 17.
|
Matsuzaki, T.,
T. Suzuki,
K. Fujikura, and K. Takata.
1997.
Nuclear staining for laser confocal microscopy.
Acta Histochem. Cytochem.
30:309-314.
|
| 18.
|
Moreno, M.,
A. Segura, and F. Garcia-Olmedo.
1994.
Pseudothionin-St1, a potato peptide active against potato pathogens.
Eur. J. Biochem.
223:135-139[Medline].
|
| 19.
|
Osborn, R. W.,
G. W. De Samblanx,
K. Thevissen,
I. Goderis,
S. Torrekens,
F. Van Leuven,
S. Attenborough,
S. B. Rees, and W. F. Broekaert.
1995.
Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae.
FEBS Lett.
368:257-262[Medline].
|
| 20.
| Osborn, R. W., and K. Thevissen. 1997. Unpublished data.
|
| 21.
|
Penninckx, I. A.,
K. Eggermont,
F. R. Terras,
B. P. Thomma,
G. W. De Samblanx,
A. Buchala,
J. P. Metraux,
J. M. Manners, and W. F. Broekaert.
1996.
Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.
Plant Cell
8:2309-2323[Abstract].
|
| 22.
|
Redman, D. G., and D. Fisher.
1969.
Purothionin analogues from barley flour.
J. Sci. Food Agric.
20:427-432.
|
| 23.
|
Roth, B.,
M. Poot,
S. Yue, and P. Millard.
1997.
Bacterial viability and antibiotic susceptibility testing with SYTOX Green nucleic acid stain.
Appl. Environ. Microbiol.
63:2421-2431[Abstract].
|
| 24.
|
Schmitt, M. J., and P. Compain.
1995.
Killer-toxin-resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor.
Arch. Microbiol.
164:435-443[Medline].
|
| 25.
|
Tejuca, M.,
M. D. Serra,
M. Ferreras,
M. E. Lanio, and G. Menestrina.
1996.
Mechanism of membrane permeabilization by sticholysin I, a cytolysin isolated from the venom of the sea anemone Stichodactyla helianthus.
Biochemistry
35:14947-14957[Medline].
|
| 26.
|
Terras, F. R.,
H. M. Schoofs,
M. F. De Bolle,
F. Van Leuven,
S. B. Rees,
J. Vanderleyden,
B. P. Cammue, and W. F. Broekaert.
1992.
Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.
J. Biol. Chem.
267:15301-15309[Abstract/Free Full Text].
|
| 27.
|
Terras, F. R. G.,
H. M. E. Schoofs,
K. Thevissen,
R. W. Osborn,
J. Vanderleyden,
B. P. A. Cammue, and W. F. Broekaert.
1993.
A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species.
FEBS Lett.
316:233-240[Medline].
|
| 28.
|
Thevissen, K.,
A. Ghazi,
G. W. De Samblanx,
C. Brownlee,
R. W. Osborn, and W. F. Broekaert.
1996.
Fungal membrane responses induced by plant defensins and thionins.
J. Biol. Chem.
271:15018-15025[Abstract/Free Full Text].
|
| 29.
|
Thevissen, K.,
R. W. Osborn,
D. P. Acland, and W. F. Broekaert.
1997.
Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes.
J. Biol. Chem.
272:32176-32181[Abstract/Free Full Text].
|
| 30.
| Thevissen, K., R. W. Osborn, D. P. Acland, and
W. F. Broekaert. Specific binding sites for an antifungal
plant defensin from dahlia (Dahlia merckii) on fungal cells
are required for antifungal activity. Submitted for publication.
|
| 31.
|
Thomma, B. P. H. J., and W. F. Broekaert.
1998.
Tissue-specific expression of plant defensin genes PDF2.1 and PDF2.2 in Arabidopsis thaliana.
Plant Physiol. Biochem.
36:533-537.
|
| 32.
|
van Belkum, M. J.,
J. Kok,
G. Venema,
H. Holo,
I. F. Nes,
W. N. Konings, and T. Abee.
1991.
The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner.
J. Bacteriol.
173:7934-7941[Abstract/Free Full Text].
|
| 33.
|
van Oss, C. J.
1997.
Hydrophobicity and hydrophilicity of biosurfaces.
Curr. Opin. Coll. Interf. Sci.
2:503-512.
|
| 34.
|
Veldhuis, M. J. W.,
T. L. Cucci, and M. E. Sieracki.
1997.
Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications.
J. Phycol.
33:527-541.
|
Applied and Environmental Microbiology, December 1999, p. 5451-5458, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Krishnakumari, V., Rangaraj, N., Nagaraj, R.
(2009). Antifungal Activities of Human Beta-Defensins HBD-1 to HBD-3 and Their C-Terminal Analogs Phd1 to Phd3. Antimicrob. Agents Chemother.
53: 256-260
[Abstract]
[Full Text]
-
Gelhaus, C., Jacobs, T., Andra, J., Leippe, M.
(2008). The Antimicrobial Peptide NK-2, the Core Region of Mammalian NK-Lysin, Kills Intraerythrocytic Plasmodium falciparum. Antimicrob. Agents Chemother.
52: 1713-1720
[Abstract]
[Full Text]
-
Hagen, S., Marx, F., Ram, A. F., Meyer, V.
(2007). The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi. Appl. Environ. Microbiol.
73: 2128-2134
[Abstract]
[Full Text]
-
Zakrzewska, A., Boorsma, A., Delneri, D., Brul, S., Oliver, S. G., Klis, F. M.
(2007). Cellular Processes and Pathways That Protect Saccharomyces cerevisiae Cells against the Plasma Membrane-Perturbing Compound Chitosan. Eukaryot Cell
6: 600-608
[Abstract]
[Full Text]
-
Lockshon, D., Surface, L. E., Kerr, E. O., Kaeberlein, M., Kennedy, B. K.
(2007). The Sensitivity of Yeast Mutants to Oleic Acid Implicates the Peroxisome and Other Processes in Membrane Function. Genetics
175: 77-91
[Abstract]
[Full Text]
-
Munoz, A., Lopez-Garcia, B., Marcos, J. F.
(2006). Studies on the Mode of Action of the Antifungal Hexapeptide PAF26. Antimicrob. Agents Chemother.
50: 3847-3855
[Abstract]
[Full Text]
-
Makovitzki, A., Avrahami, D., Shai, Y.
(2006). Ultrashort antibacterial and antifungal lipopeptides. Proc. Natl. Acad. Sci. USA
103: 15997-16002
[Abstract]
[Full Text]
-
Rosenfeld, Y., Barra, D., Simmaco, M., Shai, Y., Mangoni, M. L.
(2006). A Synergism between Temporins toward Gram-negative Bacteria Overcomes Resistance Imposed by the Lipopolysaccharide Protective Layer. J. Biol. Chem.
281: 28565-28574
[Abstract]
[Full Text]
-
Mendieta, J. R., Pagano, M. R., Munoz, F. F., Daleo, G. R., Guevara, M. G.
(2006). Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization. Microbiology
152: 2039-2047
[Abstract]
[Full Text]
-
Zakrzewska, A., Boorsma, A., Brul, S., Hellingwerf, K. J., Klis, F. M.
(2005). Transcriptional Response of Saccharomyces cerevisiae to the Plasma Membrane-Perturbing Compound Chitosan. Eukaryot Cell
4: 703-715
[Abstract]
[Full Text]
-
Park, C., Bennion, B., Francois, I. E. J. A., Ferket, K. K. A., Cammue, B. P. A., Thevissen, K., Levery, S. B.
(2005). Neutral glycolipids of the filamentous fungus Neurospora crassa: altered expression in plant defensin-resistant mutants. J. Lipid Res.
46: 759-768
[Abstract]
[Full Text]
-
Mangoni, M. L., Saugar, J. M., Dellisanti, M., Barra, D., Simmaco, M., Rivas, L.
(2005). Temporins, Small Antimicrobial Peptides with Leishmanicidal Activity. J. Biol. Chem.
280: 984-990
[Abstract]
[Full Text]
-
Bulmer, M. S., Crozier, R. H.
(2004). Duplication and Diversifying Selection Among Termite Antifungal Peptides. Mol Biol Evol
21: 2256-2264
[Abstract]
[Full Text]
-
Spelbrink, R. G., Dilmac, N., Allen, A., Smith, T. J., Shah, D. M., Hockerman, G. H.
(2004). Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant Defensins. Plant Physiol.
135: 2055-2067
[Abstract]
[Full Text]
-
Yenugu, S., Hamil, K. G., Radhakrishnan, Y., French, F. S., Hall, S. H.
(2004). The Androgen-Regulated Epididymal Sperm-Binding Protein, Human {beta}-Defensin 118 (DEFB118) (Formerly ESC42), Is an Antimicrobial {beta}-Defensin. Endocrinology
145: 3165-3173
[Abstract]
[Full Text]
-
Luque-Ortega, J. R., Martinez, S., Saugar, J. M., Izquierdo, L. R., Abad, T., Luis, J. G., Pinero, J., Valladares, B., Rivas, L.
(2004). Fungus-Elicited Metabolites from Plants as an Enriched Source for New Leishmanicidal Agents: Antifungal Phenyl-Phenalenone Phytoalexins from the Banana Plant (Musa acuminata) Target Mitochondria of Leishmania donovani Promastigotes. Antimicrob. Agents Chemother.
48: 1534-1540
[Abstract]
[Full Text]
-
Thevissen, K., Warnecke, D. C., Francois, I. E. J. A., Leipelt, M., Heinz, E., Ott, C., Zahringer, U., Thomma, B. P. H. J., Ferket, K. K. A., Cammue, B. P. A.
(2004). Defensins from Insects and Plants Interact with Fungal Glucosylceramides. J. Biol. Chem.
279: 3900-3905
[Abstract]
[Full Text]
-
Oberparleiter, C., Kaiserer, L., Haas, H., Ladurner, P., Andratsch, M., Marx, F.
(2003). Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli. Antimicrob. Agents Chemother.
47: 3598-3601
[Abstract]
[Full Text]
-
Veronese, P., Ruiz, M. T., Coca, M. A., Hernandez-Lopez, A., Lee, H., Ibeas, J. I., Damsz, B., Pardo, J. M., Hasegawa, P. M., Bressan, R. A., Narasimhan, M. L.
(2003). In Defense against Pathogens. Both Plant Sentinels and Foot Soldiers Need to Know the Enemy. Plant Physiol.
131: 1580-1590
[Full Text]
-
Theis, T., Wedde, M., Meyer, V., Stahl, U.
(2003). The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization. Antimicrob. Agents Chemother.
47: 588-593
[Abstract]
[Full Text]
-
Chicharro, C., Granata, C., Lozano, R., Andreu, D., Rivas, L.
(2001). N-Terminal Fatty Acid Substitution Increases the Leishmanicidal Activity of CA(1-7)M(2-9), a Cecropin-Melittin Hybrid Peptide. Antimicrob. Agents Chemother.
45: 2441-2449
[Abstract]
[Full Text]
-
Selitrennikoff, C. P.
(2001). Antifungal Proteins. Appl. Environ. Microbiol.
67: 2883-2894
[Full Text]
-
Thevissen, K., Cammue, B. P. A., Lemaire, K., Winderickx, J., Dickson, R. C., Lester, R. L., Ferket, K. K. A., Van Even, F., Parret, A. H. A., Broekaert, W. F.
(2000). A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii). Proc. Natl. Acad. Sci. USA
10.1073/pnas.160077797v1
[Abstract]
[Full Text]
-
Thevissen, K., Cammue, B. P. A., Lemaire, K., Winderickx, J., Dickson, R. C., Lester, R. L., Ferket, K. K. A., Van Even, F., Parret, A. H. A., Broekaert, W. F.
(2000). A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii). Proc. Natl. Acad. Sci. USA
97: 9531-9536
[Abstract]
[Full Text]
Top
Abstract
Introduction
