Fungal Genetics and Biology 46 (2009) 585–594
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Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi
Chitosan permeabilizes the plasma membrane and kills cells
of Neurospora crassa in an energy dependent manner
J. Palma-Guerrero a,b, I.-C. Huang a, H.-B. Jansson b, J. Salinas b, L.V. Lopez-Llorca b, N.D. Read a,*
a
Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK
Laboratory of Plant Pathology, Multidisciplinary Institute for Environmental Studies (MIES) Ramón Margalef, Department of Marine Sciences and Applied Biology,
University of Alicante, 03080 Alicante, Spain
b
a r t i c l e
i n f o
Article history:
Received 17 January 2009
Accepted 26 February 2009
Available online 21 April 2009
Keywords:
Aequorin
Chitosan
Conidium
Conidial anastomosis tube
Endocytosis
Fungicide
Hyphal fusion
Live-cell imaging
Neurospora crassa
Spore germination
a b s t r a c t
Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its
mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma
membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability
stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization
and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells
was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ
tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia
being the most sensitive.
Crown Copyright ! 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction
Chitosan is a polymer of b-1,4-glucosamine subunits, and is a
partly deacetylated form of chitin (Rabea et al., 2003). It is not toxic
to mammals (Dodane and Vilivalam, 1998; Lee et al., 2004) and
elicits plant defense mechanisms (Ait Barka et al., 2004; Benhamou
et al., 1994; Lafontaine and Benhamou, 1996; Trotel-Aziz et al.,
2006). Chitosan displays antibiotic activity against bacteria (Liu
et al., 2001, 2004; Tikhonov et al., 2006) and fungi (Bautista-Banos
et al., 2006; Bell et al., 1998; Laflamme et al., 1999; Palma-Guerrero et al., 2008; Park et al., 2002; Plascencia-Jatomea et al., 2003),
and has been reported to damage the plasma membranes of both
bacteria (Liu et al., 2004) and the yeast Saccharomyces cerevisiae
(Zakrzewska et al., 2005). The deletion of genes encoding proteins
that are involved in maintaining plasma membrane integrity was
found to increase the sensitivity to chitosan (Zakrzewska et al.,
2007). Plasma membrane damage has also been suggested to explain the fungicidal effects of chitosan on filamentous fungi (El
* Corresponding author. Fax: +44 131 650 5392.
E-mail address: [email protected] (N.D. Read).
Ghaouth et al., 1992; Laflamme et al., 1999). The permeabilization
of the plasma membrane by chitosan has been proposed to be
caused by the interaction of the positive amino groups of chitosan
with the negative charges on phospholipids (Liu et al., 2004). Recently, rhodamine-labeled chitosan was found to be taken up by
conidia of plant pathogenic and nematophagous fungi in an energy
dependent manner and not by passive diffusion (Palma-Guerrero
et al., 2008). Previously the endocytic marker FM4-64 had been
shown to be internalized by conidia of Magnaporthe grisea in an energy dependent manner (Atkinson et al., 2002), which suggests
that chitosan may also be endocytically internalized.
The aim of the present study was first, to test the hypothesis
that chitosan is endocytically internalized by fungal cells, and second, to obtain further insights into the mode of antifungal action
by chitosan. For this purpose we used Neurospora crassa, a species
in which endocytosis has been previously analyzed (Fischer-Parton
et al., 2000; Read and Hickey, 2001; Read and Kalkman, 2003) and
which is particularly amenable to live-cell analysis using confocal
microscopy (e.g. Hickey et al., 2005). Our results show that chitosan enters and kills cells after plasma membrane permeabilization
by an unknown energy dependent mechanism that does not involve endocytosis. We have also found that conidia, germ tubes,
and vegetative hyphae exhibit differential sensitivity to the fungicidal effects of chitosan.
1087-1845/$ - see front matter Crown Copyright ! 2009 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2009.02.010
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2. Materials and methods
2.1. Strains and culture conditions
The N. crassa wild-type strain 74-OR23-IVA (FGSC #2489), and
the N. crassa H1-GFP strain (N2283, Freitag et al., 2004) were grown
and maintained on solid Vogel’s agar medium (Davis, 2000). A transformed strain of N. crassa expressing codon-optimized aequorin
(strain #272, Fungal Cell Biology Group, University of Edinburgh,
UK; Nelson et al., 2004; Zelter, 2004) was stored as a stock culture
on solid Vogel’s agar medium containing 200 lg ml!1 hygromycin
B, but was grown on solid Vogel’s medium lacking hygromycin B
for the production of the conidia used in luminometry experiments.
2.2. Chemicals and dyes
Chitosan (T8s) with a molecular weight of 70 KDa and exhibiting 79.6% deacetylation was obtained from Marine BioProducts
GmbH (Bremerhaven, Germany). Chitosan was dissolved in
0.25 mol l!1 HCl and the pH adjusted to 5.6 with 1 mol l!1 NaOH.
The resulting solution was dialyzed for salt removal, and the dialyzed chitosan was autoclaved at 120 "C for 20 min (Palma-Guerrero et al., 2008).
Rhodamine-labeled chitosan (rhodamine-T8s chitosan) was
kindly provided by Dr. V. Tikhonov (Laboratory of Physiologically
Active Biopolymers, A.N. Nesmeyanov Institute of Organoelement
Compounds, Russian Academy of Sciences, Moscow, Russia). After
dialysis, the labeled chitosan was filtered through 3 KDa ultramembranes in an Amicon Cell (Omega, Pall Corporation, Ann Arbor, MI, USA) and sterilized by filtration through a 0.2 lm poresize syringe filter (Albet, Barcelona, Spain).
Sodium azide and the cell viability dyes, fluorescein diacetate
(FDA) and propidium iodide (PI), were obtained from Sigma (St.
Louis, MO, USA). The membrane selective fluorescent dye, FM464, was obtained from Invitrogen (Eugene, OR, USA). Stock solutions of FDA (1 mg ml!1 in acetone), PI (1 mg ml!1 in H2O) and
FM4-64 (10 mg ml!1 in DMSO) were all diluted in distilled H2O
and then added to the conidial inocula at final concentrations of
5 lg ml!1, 5 lg ml!1, and 5 lM, respectively.
2.3. Quantitation of conidial germination, conidial lysis and CAT fusion
Conidia were collected from 4 to 5 day old wild type cultures by
adding 1 ml sterile distilled water and removing the conidial suspension with a pipette. The conidia were counted using a haemocytometer, diluted to the appropriate concentration, and
immediately used in the bioassays. Conidial germination, conidial
lysis and conidial anastomosis tube (CAT) fusion assays were carried out in 8-well slide culture chambers (Nalge Nunc International, Rochester, NY). Each well was filled with 200 ll of
conidial suspension at a final concentration of 106 conidia ml!1 in
potato dextrose broth (PDB) (Becton Dickinson and Company,
Sparks, MD, USA) with chitosan at the appropriate final concentration. PDB (2.5 g l!1) was used for the conidial germination and
conidial lysis assays. PDB was replaced with liquid Vogel’s medium
(Davis, 2000) diluted 100 times for the CAT fusion assay. After
incubation at 24 "C in continuous light, the number of non-lysed
conidia were counted (for conidial lysis assays) or the percentage
conidia that had not lysed and had germinated were quantified
(for conidial germination assays). Percentage germination was defined as the percentage of conidia possessing one or more germ
tubes and/or CATs. A 20" dry or a 60" water immersion plan
apo objective with differential interference contrast (DIC) optics
on an inverted TE2000E microscope (Nikon, Kingston-UponThames, United Kingdom) was used in the germination and lysis
assays. CAT fusion was only assessed with the 60" objective on
the inverted microscope and CAT fusion was quantified as the percentage of conidia/conidial germlings involved in fusion. Three
wells per treatment, with ten fields of view per well (each containing 100–300 conidia), were assessed for each of the three experimental assays, and each experiment was performed twice.
2.4. Monitoring rhodamine-chitosan internalization
Conidia were collected as described in Section 2.3 and were
treated with 0.1 mg ml!1 rhodamine-chitosan for different times
at room temperature (#22 "C). The conidia were washed three
times with distilled water by centrifugation at 19,300g for 5 min
to remove unbound rhodamine-chitosan. The resultant conidial
suspension was then dispensed as 30–50 ll droplets onto glass
coverslips, and conidia were immobilized by the inverted agar
block method (Hickey et al., 2005) and imaged by confocal microscopy (Section 2.8) with a 100" oil immersion objective.
2.5. Staining with fluorescent probes
Staining of conidia with FM4-64 or PI was carried out by adding
FM4-64 (at a final concentration of 5 lM) or PI (at a final concentration of 5 lg ml!1) to a conidial suspension prepared as described in
Section 2.3. For the chitosan treatment, the conidia (to which FM464 or PI had been added) were treated with chitosan at a final concentration of 0.1 mg ml!1. The conidial suspensions were inoculated, immobilized and imaged as described in Section 2.4.
2.6. Cell viability tests
A combination of FDA and PI was used to check cell viability
after conidia and conidial germlings were treated with chitosan.
Wells of the 8-welled culture chambers (Section 2.3) were filled
with 100 ll of conidial suspension at a final concentration (after
dye addition) of 106 conidia ml!1. FDA and PI were added to the
conidial suspension at a final concentration of 5 lg ml!1. Once
the culture chamber was mounted on the confocal microscope
stage, 100 ll of chitosan was added at a final concentration of
0.1 mg ml!1. Samples were immediately imaged using a 40" dry
or a 60" water immersion objective, and images were captured
every 30 s for 12 min. For the germling viability tests, 100 ll of
conidial suspension in 2.5 g l!1 PDB was first incubated in each
well of the 8-welled culture chamber for 6 h at 24 "C in continuous
light. FDA, PI and chitosan were added as described in Section 2.5.
Ungerminated conidia and 6 h old conidial germlings were imaged
by confocal microscopy (Section 2.8) with a 20" dry or a 60"
water immersion plan apo objective, and an image was captured
every min for 1 h. The percentages of live and dead conidia or
germlings were recorded at each time point and the percentage
of live cells was determined. Three wells per treatment were recorded (100–300 conidia per field of view, 10 fields of view per
well) and the experiment was performed twice.
FM4-64 was used to check the viability of vegetative hyphae after
chitosan treatment. The wells containing 100 ll conidial suspension
at a final concentration of 106 conidia ml!1 in 2.5 g l!1 PDB were
incubated for 18 h at 24 "C in continuous light. FM4-64 was added
to the vegetative hyphae at a final concentration of 5 lM. Chitosan
was added at a final concentration of 0.1 mg ml!1 and samples were
immediately imaged by confocal microscopy (Section 2.8) with a
20" objective, and an image captured every min for 1 h.
2.7. Inhibition of energy dependent processes
Sodium azide and low temperature (4 "C) were used to inhibit ATP
production and thus energy dependent processes (Atkinson et al.,
2002; Hoffmann and Mendgen, 1998). Conidia or conidial germlings
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were either pre-treated for 30 min with 75 mM sodium azide or kept
at 4 "C for 30 min, before treating them with rhodamine-chitosan,
chitosan, the fluorescent dyes, or a combination of chitosan and the
dyes. Pre-treatment with 70% ethanol for 30 min was used to kill conidia to assess rhodamine-chitosan uptake by dead cells.
2.8. Confocal microscopy
Confocal laser scanning microscopy was performed using a Radiance 2100 system, equipped with argon ion and green HeNe lasers
(Bio-Rad Microscience), which was mounted on a Nikon TE2000U
Eclipse inverted microscope. Rhodamine-chitosan was imaged with
excitation at 543 nm and fluorescence detection at >560 nm. FM464 was imaged by excitation at 488 nm and fluorescence detection
at >560 nm. When rhodamine-chitosan and FM4-64 were combined
and imaged simultaneously, excitation was at 543 nm and fluorescence detection at 560–620 nm (for rhodamine-chitosan) and
>650 nm (for FM4-64). FDA and PI were detected simultaneously
using excitation at 488 nm and detection at 500–530 nm (FDA)
and >560 nm (PI). GFP and PI were detected simultaneously with
the same filter settings used for FDA and PI. The laser intensity
and laser scanning were reduced to a minimum to reduce phototoxic effects. Simultaneous DIC images were captured with a transmitted light detector. Kalman filtering (n = 1 or n = 2) was used to
improve the signal-to-noise ratio of images. Images were captured
with a laser scan speed of 166 lines per sec at a resolution of 512
by 512 pixels. Confocal images were captured with Lasersharp
2000 software (version 5.1; Bio-Rad Microscience) and processed
using Image J (version 1.38x for Mac Os; freeware) and Adobe Photoshop (version 10.0; Adobe Systems, Inc.) software.
2.9. Luminometry
106 conidia ml!1 of the strain expressing codon-optimized
aequorin (Nelson et al., 2004; Zelter, 2004) were incubated in
2.5 g l!1 PDB medium with 0.68 mM CaCl2 (Sigma, St. Louis, MO,
USA) and 10 lM coelenterazine (Biosynth, Staad, Switzerland)
added. The coelenterazine was initially dissolved in methanol and
subsequently diluted in 2.5 g l!1 PDB (Nelson et al., 2004). 90 ll of
the conidial suspension was dispensed into each of the individual
flat-bottomed wells of a white 96-well plate (Fisher scientific,
Loughborough, Leicestershire, UK). Each plate was covered with aluminum foil and incubated: (a) at 4 "C for 6 h in the dark (for the analysis of ungerminated conidia), (b) at 24 "C in the dark for 6 h (for the
analysis of conidial germlings), or (c) at 24 "C in the dark for 18 h (for
the analysis of vegetative hyphae). Luminometry was performed
using a Berthold LB96P plate luminometer equipped with two injectors. In some experiments, 100 ll chitosan, diluted to 0.2 mg ml!1 in
2.5 g l!1 PDB with 0.68 mM CaCl2, was injected into wells containing
the fungal material (the final concentration of chitosan was
0.1 mg ml!1). Control injections omitted the chitosan. In other
experiments, 10 ll sodium azide (at a final concentration of
75 mM) was pipetted into the wells containing fungal material
30 min before the injection of the chitosan. Controls for these experiments involved adding 10 ll of distilled H2O per well. Measurements from six wells for each treatment were performed in each
experiment, and each experiment was performed three times.
3. Results
3.1. Chitosan inhibits conidial germination and conidial germling
fusion
To assess the effects of different concentrations of chitosan on
conidial germination and conidial germling fusion via conidial
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anastomosis tubes (CATs) (Roca et al., 2005; Read et al., in press),
liquid Vogel’s medium (the standard growth medium used for N.
crassa, Davis, 2000), was initially used. However, the addition of
chitosan was found to cause visible precipitation in Vogel’s medium when the medium was used at its normal concentration. An
alternative medium, potato dextrose broth (PDB), in which chitosan-induced precipitation was not evident, was used. After 6 h at
24 "C in PDB 2.5 g l!1, 98.8% (sD ± 0.1) of untreated conidia had germinated (Fig. 1A). Conidial germination was completely inhibited
at chitosan concentrations of 0.01 or 0.1 mg ml!1 and none of these
conidia germinated after 24 h. After 6 h, conidia treated with 0.01
or 0.1 mg ml!1 chitosan were shrunken and highly vacuolated
(Fig. 1B). Treatment with lower concentrations of chitosan showed
that the 50% inhibitory concentration (IC50) of chitosan for conidial
germination in PDB was #7.5 lg ml!1.
Unexpectedly we discovered that CAT fusion was greatly inhibited in PDB and only 4.0% sD ± 1.26 of conidial germlings underwent fusion. To assess the effects of chitosan on CAT fusion we
diluted Vogel’s medium 100 times to a concentration at which
no signs of chitosan precipitation were observed (not shown). After
6 h at 24 "C in 100" diluted Vogel’s medium, 95.0% (sD ± 0.3) of untreated conidia had germinated (produced a germ tube and/or a
CAT) and 48.0% (sD ± 1.2) of conidial germlings were involved in
CAT fusion (Fig. 1C). The IC50 values of chitosan for conidial germination and CAT fusion in this medium were both #0.25 lg ml!1
and thus neither process was more sensitive to chitosan than the
other.
3.2. Rhodamine-labeled chitosan uptake is energy dependent but not
by endocytosis
To monitor the internalization of chitosan by living cells, chitosan fluorescently labeled with rhodamine was used. Rhodaminechitosan was detected inside conidia within 30 min of addition
(Fig. 2A). Treatment of conidia of M. grisea with the mitochondrial
ATP synthetase inhibitor sodium azide, or incubation at low temperature (4 "C), were previously shown to inhibit energy dependent internalization of endocytic marker dyes (Atkinson et al.,
2002). Both treatments were also found to inhibit the uptake of
rhodamine-chitosan by conidia of N. crassa. Consequently, rhodamine-chitosan fluorescence was only detected bound to what
seemed to be the conidial cell wall (Fig. 2B and C). However, the
localization of rhodamine-chitosan in the putative cell wall was
non-uniform around conidia (Figs. 2A–C, and 3B). Conidia that
had taken up rhodamine-chitosan looked unhealthy because their
cytoplasm had a ’granular’ and partially vacuolated appearance
when imaged with DIC optics (Fig. 2A). This contrasted with untreated conidia in which the cytoplasm had a ’smoother’ appearance (Fig. 2D). The conidia that had been subjected to the
inhibitory treatments all had a similar healthy appearance to the
untreated conidia (Fig. 2B–D). When rhodamine-chitosan was applied to conidia that had been killed with 70% ethanol, fluorescence
was detected within the conidia, and the DIC image shows that the
cytoplasm of the killed cells was granular, similar to rhodaminechitosan-treated conidia (Fig. 2E). The pattern of rhodamine-chitosan localization in the killed conidia (Fig. 2E) was different from
that in the untreated living conidia (Fig. 2A).
To determine if rhodamine-chitosan was entering cells by endocytosis, its uptake was compared with that of the endocytic marker
FM4-64 (Fischer-Parton et al., 2000; Hickey et al., 2005). Rhodamine-chitosan and FM4-64 showed markedly different patterns
of internalization. Five minutes after applying FM4-64, fluorescence was detected in the conidial plasma membrane and in small
endosome-like structures. Between 15 and 60 min more organelles, including larger ones, became increasingly stained (Fig. 3A).
This staining pattern is similar to the sequential staining of
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Fig. 1. (A) Conidial germlings in 0.25% PDB after 6 h at 24 "C in continuous light.
Untreated control without chitosan. Note that although a number of germ tubes are
touching each other, none of the conidial germlings have undergone fusion. (B)
Dead conidia after treatment with 0.1 mg ml!1 chitosan in 0.25% PDB for 6 h at
24 "C in continuous light. (C) Conidial germlings in 0.01% Vogel’s medium after 6 h
at 24 "C in continuous light. Note that a number of conidial germlings have fused
(asterisks) by means of conidial anastomosis tubes. Bar = 10 lm.
Fig. 2. Conidia treated with 0.1 mg ml!1 rhodamine-chitosan for 30 min. Fluorescence images on the left and DIC images of same conidia on the right. (A) Conidia
without any pre-treatment. (B) Conidia pre-treated with 75 mM sodium azide for
30 min. (C) Conidia pre-treated at 4 "C for 30 min. (D) Conidia to which rhodaminechitosan has not been added. (E) Dead conidia after treatment with 70% ethanol for
30 min. Note that rhodamine-chitosan has not been taken up by conidia after pretreatments that inhibit ATP production (B and C). Bar = 5 lm.
J. Palma-Guerrero et al. / Fungal Genetics and Biology 46 (2009) 585–594
organelles in conidia of M. grisea observed by Atkinson et al.
(2002). Rhodamine-chitosan, on the other hand, did not seem to
stain the plasma membrane but only stained some regions of the
conidial surface, and seemed to be primarily taken up into the
cytoplasm in which they showed negative staining of organelles
(Fig. 3B). Rhodamine-chitosan could be detected inside conidia
within 5 min of application (Fig. 3B). When FM4-64 was
applied to rhodamine-chitosan treated conidia, it showed little
co-localization with rhodamine fluorescence (data not shown).
3.3. Chitosan permeabilizes cells in an ATP-dependent manner
The non-specific localization of rhodamine labeled chitosan
within conidia (Fig. 3B) suggests that chitosan itself permeabilizes
the plasma membrane allowing it to enter the cytoplasm. To confirm this, experiments were performed with unlabeled chitosan in
combination with the endocytic marker dye, FM4-64 (Fischer-Parton et al., 2000; Hickey et al., 2005). FM4-64 uptake by conidia was
initially shown to be energy dependent by being blocked by sodium azide treatment (Fig. 4C) or incubation at 4 "C (Fig. 4E), as
previously reported for conidia of M. grisea (Atkinson et al.,
2002). Treatment with unlabeled chitosan for 30 min, however,
produced a different pattern of FM4-64 staining (Fig. 4B) compared
with the untreated control (Fig. 4A). FM4-64 staining after chitosan
treatment was much more intense suggesting that the dye had diffused into the cells and bound non-selectively to membranous
organelles. This pattern of FM4-64 staining has been described previously in permeabilized fungal cells (Read and Roca, 2006). Sodium azide treatment or incubation at 4 "C prevented FM4-64
uptake by chitosan-treated-conidia (Fig. 4D and F) indicating that
chitosan induced permeabilization of the plasma membrane is
ATP dependent.
3.4. Chitosan induces rapid Ca2+ uptake by conidia and conidial
germlings
N. crassa, like other eukaryotic cells, normally maintains its
cytosolic free Ca2 ([Ca2+]c) concentration at a very low level (typically 50–100 nM) (Miller et al., 1990). This contrasts markedly
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with the extracellular free Ca2 concentration which, in the media
used in the experiments, is several orders of magnitude higher.
Eukaryotic cells are able to maintain an extremely steep free
Ca2+ gradient across the plasma membrane by means of a tightly
regulated [Ca2+]c homeostatic mechanism involving Ca2+ being actively transported up the Ca2+ gradient out of the cytosol either
into the extracellular medium and/or into intracellular Ca2+ storage organelles. Permeabilization of the plasma membrane can
dramatically upset [Ca2+]c homeostasis in the cell resulting in
the [Ca2+]c increasing. To analyze the influence of chitosan on
membrane permeabilization we measured changes in luminescence in a transformant expressing the bioluminescent [Ca2+]c reporter, aequorin (Nelson et al., 2004; Zelter, 2004). However, in
these experiments we did not convert the aequorin luminescence
into [Ca2+]c concentration because the extremely high levels of
aequorin light emission following treatment with 0.1 mg ml!1
chitosan reflected increases in [Ca2+]c concentration (>10 lM) that
were too high to measure with normal aequorin (Cobbold and
Lee, 1991).
Previously it has been found that ungerminated conidia of N.
crassa contain much lower levels of aequorin compared with 6 h
old germlings and 18 h old vegetative hyphae (Marris, 2007). This
is the reason why the amount of light emission (measured in relative light units [RLUs]) is so low in Fig. 5A compared with Fig. 5B
and C. A common and easily applied stimulus to fungal cells in
an individual well of a multiwell inside the luminometer is
mechanical perturbation. This involves injecting onto the cells in
each well 100 ll of growth medium which is iso-osmotic to the
medium that the cells are incubated in (Nelson et al., 2004). When
the conidial germlings and vegetative hyphae were stimulated in
this way, a small transient increase in aequorin luminescence
(amplitude = 1400–2200 RLUs) which reached its maximum
amplitude within 2 s of stimulation (Fig. 5B and C). In contrast,
no significant change in luminescence, and thus no response, was
observed when the ungerminated conidia were subjected to
mechanical perturbation (Fig. 5A). The transient increase in
[Ca2+]c in conidial germlings and vegetative hyphae (Fig. 5B and
C) will be part of the response when injecting the chitosan solution
onto these cells.
Fig. 3. Comparison of a time course of staining with 5 lM FM4-64 (A) with the uptake of 0.1 mg ml!1 rhodamine-chitosan uptake (B) by conidia. Note that FM4-64 and
rhodamine-chitosan exhibit different patterns of localization within the conidia. Bar = 5 lm.
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Treating conidia and conidial germlings with 0.1 mg ml!1 chitosan resulted in very large transient increases in aequorin luminescence (Fig. 5A and B). With the vegetative hyphae, however, a very
different result was obtained. In this case, there was an initial transient increase in aequorin luminescence (probably mainly a result
of mechanical stimulation) which was followed by a much longer
transient increase in aequorin luminescence (Fig. 5C). These results
indicate that chitosan significantly permeabilizes the plasma
membranes of both conidia and conidial germlings causing rapid
Ca2+ uptake but that the permeabilization of vegetative hyphae occurs much more slowly and too a lesser extent.
Pre-treating all three cell types with 75 mM sodium azide in
each case resulted in very significant inhibition of the increases
in aequorin luminescence following treatment with 0.1 mg ml!1
chitosan (Fig. 5A–C). With ungerminated conidia, no significant increase in luminescence could be detected following azide treatment (Fig. 5A). With conidial germlings and vegetative hyphae
treated with sodium azide, however, there was an initial transient
increase in aequorin luminescence (again probably mainly a result
of mechanical stimulation), which was followed by a much longer
transient increase in aequorin luminescence. This longer luminescence transient had a larger maximum amplitude in conidial germlings compared with vegetative hyphae. However, direct
comparison of the luminescence measured with the different cell
types was not possible because the amount of aequorin synthesized per unit weight of the conidal germlings and vegetative hyphae was not determined. These results indicate that: (a) the
rapid Ca2+ uptake in conidial germlings resulting from chitosan
injection is mostly ATP dependent but that azide induces a secondary increase in Ca2+ uptake that is not ATP dependent and (b) the
longer luminescent transient induced by chitosan in vegetative hyphae is mostly ATP dependent.
3.5. The rate of cell death is cell type specific
A combination of fluorescein diacetate (FDA) and propidium iodide (PI) was used to assess cell viability after applying chitosan.
FDA is taken up by living cells and is hydrolyzed to fluorescein,
which fluoresces green. PI is taken up by dead cells with damaged
plasma membranes and fluoresces red (Oparka and Read, 1994;
Hickey et al., 2005). By simultaneously imaging cells in the presence
of FDA and PI after applying chitosan it was possible to assess which
cells were living (green) and which were dead (red), and determine
the time required by chitosan to kill these cells (Figs. 6–8).
Four minutes after applying 0.1 mg ml!1 chitosan to a conidial
suspension, 99.15% of the conidia were dead (Fig. 8). Most of the
Fig. 4. Chitosan permeabilizes the plasma membrane of conidia in an energy dependent manner. Conidia were stained with 5 lM FM4-64. Fluorescence images on the left
and DIC images of same conidia on the right. (A) Conidia treated with FM4-64 only (control). Note that the staining of internal organelles here is greater than at the 30 min
time point in Fig. 3A. This is because FM4-64 was applied to the conidia in different ways. In Fig. 3A it was added using the ‘inverted agar’ method (Hickey et al., 2005) while
here it was added to the conidia in suspension. (B) Conidia treated with 0.1 mg ml!1 chitosan and FM4-64 simultaneously for 30 min. Note very bright staining of internal
organelles due to permeabilization of the conidial plasma membrane. (C) Conidia pre-treated with 75 mM sodium azide for 30 min and stained with 5 lM FM4-64 for 30 min.
(D) Conidia pre-treated with 75 mM sodium azide for 30 min and treated with 0.1 mg ml!1 chitosan and FM4-64 simultaneously for 30 min. (E) Conidia pre-treated at 4 "C for
30 min and stained with FM4-64 for 30 min at 4 "C. (F) Conidia pre-treated at 4 "C for 30 min and treated with 0.1 mg ml!1 chitosan and FM4-64 simultaneously for 30 min at
4 "C. Note that no FM4-64 were internalized in (C–F) Bar = 5 lm.
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Fig. 5. Luminometry of strain expressing recombinant aequorin subjected to mechanical stimulation alone (control), treatment with 0.1 mg ml!1 chitosan on its own, and
treatment with 0.1 mg ml!1 chitosan after pre-treatment for 30 min with 75 mM sodium azide. (A) Conidia. (B) 6 h old conidial germlings. (C) 18 h old vegetative hyphae.
Values ± standard errors. Arrows indicate the points in time when mechanical perturbation by injection was applied.
Fig. 6. Chitosan-induced cell death at different times following treatment with 0.1 mg ml!1 chitosan. Living cells stained green with 5 lg ml FDA and dead cells stained red
with 5 lg ml PI. Fluorescence images on the left and DIC images of same conidia on the right. (A) Conidia after 1 min. (B) Conidia after 3 min. (C) Conidia after 7 min. (D)
Conidial germlings after 1 min. (E) Conidial germlings after 35 min. (F) Conidial germlings after 50 min. Bar = 5 lm for (A–C). Bar = 10 lm for (D–F).
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J. Palma-Guerrero et al. / Fungal Genetics and Biology 46 (2009) 585–594
Fig. 7. Nuclear breakdown after treatment of conidia of a strain in which H1-GFP
has been targeted to the nuclei. Conidia treated with 0.1 mg ml!1 chitosan in the
presence of 5 lg ml PI. Fluorescence images on the left and DIC images of same
conidia on the right. (A) Nuclei are intact and fluorescing green 1 min after chitosan
treatment. (B) H1-GFP has been lost from nuclei which have become stained with PI
3 min after chitosan treatment. Bar = 5 lm.
conidia died between 1.5 and 4 min (Figs. 6A–C, 7B and 8A). This
pattern of cell death was completely inhibited after sodium azide
pre-treatment (Fig. 8A).
When 0.1 mg ml!1 chitosan was applied to conidial germlings
most of them died between 35 and 45 min (Figs. 6D–F and 8B). Cell
death was inhibited by sodium azide pre-treatment, but not completely (Fig. 8B).
Problems and inconsistent results were encountered when
using FDA and PI with vegetative hyphae. We therefore used
FM4-64 instead because it has been shown to cause markedly
more intense staining of dead than of living hyphae (Read and
Roca, 2006). When 0.1 mg ml!1 chitosan was applied to vegetative
hyphae they started to die #20 min after application and by 40 min
they were all dead. Cell death thus occurred over a similar time
period to germlings treated with chitosan.
To monitor nuclear breakdown by chitosan, a strain in which
H1-GFP was targeted to nuclei was used in combination with PI.
The disappearance of green H1-GFP nuclear fluorescence and
appearance of red PI fluorescence was detected just after plasma
membrane permeabilization occurred and was mostly complete
within #3 min (Fig. 7).
Fig. 8. Quantitation of the time course of cell death following treatment with
0.1 mg ml!1 chitosan in the presence of 5 lg ml FDA and 5 lg ml PI. (A) conidia and
(B) 6 h old condial germlings. Values ± standard errors.
3.6. Chitosan causes conidial lysis
During the analysis of the effects of chitosan on conidial germination we observed that the number of conidia at the beginning of
an experiment seemed to be lower than at the end of it. When we
quantified this we discovered that chitosan caused a concentration-dependent lysis of conidia. The IC50 for conidial lysis was
#0.0075 mg ml!1 chitosan (Fig. 9).
Fig. 9. Time course of conidal lysis in the presence of different concentrations of
chitosan. Values ± standard errors.
4. Discussion
We analyzed the mode of fungicidal action of chitosan on fungal cells using the experimental model, N. crassa, and made a
number of novel findings. First, we showed that chitosan permeabilizes the fungal plasma membrane and is internalized by fungal
cells. Both of these processes were found to be ATP dependent.
J. Palma-Guerrero et al. / Fungal Genetics and Biology 46 (2009) 585–594
Second, chitosan was found to cause the lysis of fungal spores. Finally we showed that different cell types (conidia, germ tubes
and vegetative hyphae) exhibit differential sensitivity to chitosan.
Our previous results suggested that fluorescently labeled chitosan requires ATP to enter conidia of the plant-pathogenic fungus
Fusarium oxysporum f.sp. radicis-lycopersici and the nematophagous
fungus Pochonia chlamydosporia (Palma-Guerrero et al., 2008). This
finding has been confirmed in the present study with conidia and
conidial germlings of N. crassa by using two standard treatments
to inhibit ATP production: (a) sodium azide and (b) low temperature (4 "C) (Atkinson et al., 2002; Hoffmann and Mendgen, 1998).
Thus ATP seems to be required for chitosan uptake by filamentous
fungi. Furthermore, we have shown that ATP-dependent uptake is
required to kill fungal cells.
We first tested the hypothesis that chitosan enters conidia by
the energy dependent process of endocytosis. It is now clear that
endocytosis is an important part of the vesicle trafficking network
in filamentous fungi where it serves a number of important roles
(Read and Kalkman, 2003; Fuchs and Steinberg, 2005; Peñalva,
2005; Fuchs et al., 2006; Steinberg, 2007; Sánchez-Ferrero and
Peñalva, 2008; Taheri-Talesh et al., 2008; Upadhyah and Shaw,
2008). Much of the evidence for endocytosis in filamentous fungi
has involved the use of the endocytic marker dye, FM4-64, and this
has been extensively tested with cells of N. crassa (Fischer-Parton
et al., 2000; Hickey et al., 2005; Araujo-Palomares et al., 2007;
Riquelme et al., 2007), although not previously with conidia of this
species. Nevertheless, the endocytic internalization of FM4-64 has
been well characterized during conidial germination in the plant
pathogen, M. grisea (Atkinson et al., 2002). In our study we showed
that FM4-64 is internalized in an ATP-dependent manner by conidia of N. crassa which is consistent with it being internalized by
endocytosis. However, the differences in localization of rhodamine-chitosan and FM4-64 indicate that chitosan is not internalized by endocytosis in filamentous fungi.
Using N. crassa expressing recombinant aequorin, chitosan was
found to cause permeabilization of the plasma membrane to external Ca2+ within two seconds. This resulted in a very large and rapid
Ca2+ uptake into conidia and conidial germlings, although the
kinetics of Ca2+ uptake by these two cell types was different. In
contrast, Ca2+ uptake by vegetative hyphae was less rapid and a
lower amount of Ca2+ was taken up. The pattern of chitosan-induced Ca2+ uptake is thus cell type specific. The experiments with
the sodium azide pre-treatment to inhibit ATP formation showed
that the rapid Ca2+ uptake by conidia following chitosan treatment
seemed to be completely ATP dependent, and mostly ATP dependent in conidial germlings and vegetative hyphae. The sodium
azide pre-treatment of conidial germlings induced a secondary increase in Ca2+ uptake that is not ATP dependent. It is possible that
this relates to the permeabilization of a subpopulation of germlings which were shown to die in response to chitosan + azide
treatment (Fig. 8B). The longer luminescent transient induced by
chitosan in vegetative hyphae was mostly ATP dependent. Calibration of the [Ca2+]c was not possible in the fungal cells because the
[Ca2+]c changes were so large they were out of the dynamic range
that can be measured with normal aequorin (Cobbold and Lee,
1991).
Although our results show that exogenous chitosan causes very
rapid, ATP dependent permeabilization of the plasma membrane, it
is not clear how endogenous ATP is required for this process to occur. One possibility is that after chitosan interacts with membrane
components, ATP binds to a molecule (e.g. lipid or protein) on the
cytoplasmic side of the plasma membrane, causing a conformational change which allows the formation of pores that traverse
the plasma membrane. The binding of ATP to a number of proteins
have been found to cause conformational changes which result in
regulatory activity (Traut, 1994).
593
Cell viability tests using FDA and PI that are based on the esterase activity of intact living cells and detection of plasma membrane
damage, respectively, showed that chitosan killed conidia in less
than four minutes, conidial germlings within 35–45 min, and vegetative hyphae within 40 min. Nuclear destruction, using a N. crassa H1-GFP transformant, was found to occur within #3 min, and
some conidia were found to lyse in the presence of chitosan. Neither chitosan-induced nuclear destruction nor cell lysis have been
previously demonstrated in filamentous fungi. The lower sensitivity to chitosan of germlings and hyphae than conidia may relate to
differences in the composition of their plasma membranes. For
example, different amounts of ergosterol have been found in different fungal cell types (Alvarez et al., 2007; Martin and Konopka,
2004; Van Leeuwen et al., 2008).
CAT fusion was found to be similarly sensitive to chitosan (the
IC50 in Vogel’s medium diluted 100 times was #0.25 lg ml!1.
However, during the course of this study we made the interesting
observation that CAT fusion is very much reduced when conidia
were germinated in PDB. We have also found that hyphal fusion
in the mature colony is very reduced or possibly absent when N.
crassa is grown on solid potato dextrose agar medium (Huang, I.C and Read, N.D., unpublished data). At this stage it is not clear
whether PDB contains an inhibitor or Vogel’s medium contains a
stimulator of CAT and hyphal fusion. Nevertheless, the ability to inhibit hyphal fusion from occurring by simply growing the fungus
on PDB medium will provide a very useful experimental tool to
analyze the roles of CAT and hyphal fusion in the future, and will
complement the use of hyphal fusion mutants in these studies
(Read et al., in press).
This paper helps our understanding of the mode of action of
chitosan, which has great potential as a fungicide (Rabea et al.,
2003). Further work is in progress to elucidate the mechanism by
which chitosan permeabilizes the plasma membranes of filamentous fungi.
Acknowledgments
We thank Dr. D. Blome (Marine Bioproducts GmbH, Bremerhaven, Germany) for the kind gift of the T8s chitosan used in
the experiments presented in this paper. We also thank Dr. V.
Tikhonov (A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences) for providing the rhodamine-labeled chitosan. Alexander Lichius and Dr. Pete I. Marris
(Fungal Cell Biology Group, University of Edinburgh) are greatly
acknowledged for skillful technical assistance with the confocal
microscope and luminometer, respectively. Thanks are also due
to Dr. Gabriela Roca who made the initial observation that CAT
fusion is inhibited in PDB. This study was supported by grants
from Biotechnological and Biological Research Council (BB/
E010741/1) and the Spanish Ministry of Science and Innovation
(AGL2008-00716).
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