From the Laboratoire de Mycologie Fondamentale et
Appliquée, Inserm EMI0360, Université de Lille II, and
¶ Inserm U459, Faculté de Médecine H. Warembourg,
Place Verdun, 59037 Lille Cedex, France
Received for publication, October 18, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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The surface of the pathogenic yeast Candida
albicans is coated with phospholipomannan (PLM), a
phylogenetically unique glycolipid composed of Candida albicans is part of the normal
microbial flora that colonizes the mucocutaneous surfaces of the oral
cavity, gastrointestinal tract, and vagina. The high levels of
morbidity and mortality induced by C. albicans in
hospitalized patients mean that this species is now one of the most
prominent human pathogens (1). Research is currently under way to
identify the virulence attributes of C. albicans (2) that
explain the success of this species as a human pathogen.
It has previously been shown that C. albicans, in contrast
to the closely related but nonpathogenic yeast, Saccharomyces
cerevisiae, was able to survive within macrophages (3).
Endocytosis of C. albicans by macrophages was specifically
associated with the reduced phosphorylation of ERK1/2 and the
downstream product, p90rsk, through activation of a
specific phosphatase, MKP-1 (3). Both ERK1/2 and p90rsk have
been shown to regulate survival of different cells (4 Intracellular pathogens have evolved diverse strategies to induce
(10-13) or inhibit host cell apoptosis (14-16), aiding dissemination within the host or facilitating intracellular survival (17, 18). Host
cell apoptosis is induced through different virulence mechanisms based
on either surface glycolipids (15, 19, 20) or type III secretion
proteins (21, 22). In host tissues, C. albicans may be
both intra- and extracellular (23). Macrophages undergo apoptotic cell
death after infection with C. albicans strains capable of
hyphal formation (24), and activation of caspase 3 has been observed
after endocytosis of C. albicans by neutrophils (25).
It has recently been demonstrated that the cell wall surface of
C. albicans is coated with a phylogenetically unique
molecule, phospholipomannan
(PLM),1 composed of
oligomannose residues with a unusual type of linkage and phytoceramide
(26, 27) (Fig. 1A). PLM is shed by C. albicans in
contact with macrophages (28) and displays potent activity on the
innate immune response (29).
In this study, the modulation of signals downstream from ERK1/2 and
p90rsk was investigated, with special attention to the
regulation of cell survival induced after endocytosis of C. albicans. The participation of C. albicans PLM as an
inducer of apoptosis was then determined using highly purified and well
characterized PLM. The addition of PLM to cells was found to allow
survival of the sensitive yeast S. cerevisiae. This was
associated with down-regulation of ERK1/2-dependent Bad
Ser-112 phosphorylation in host cells and alteration of cell integrity
leading to apoptosis.
Reagents and Antibodies--
All reagents were obtained from
Sigma. Specific rabbit polyclonal IgGs to the phosphorylated forms of
Bad (Ser- or Ser-136), ERK1/2, and p90rsk were purchased from
New England Biolabs (Beverly, MA). Anti-Bcl-2 rabbit polyclonal IgG was
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Horseradish peroxidase and fluorescein isothiocyanate-conjugated
anti-rabbit IgG were obtained from Southern Biotechnology
Laboratories (Birmingham, AL). Anti- Cell Culture--
The mouse macrophage-like cell line, J774
(ECACC 85011428), was derived from a tumor of a female BALB/c mouse.
Adherent cells were cultured at 37 °C in an atmosphere containing
5% CO2 in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% heat-inactivated fetal calf serum (Valbiotech,
Paris, France), 5 mM L-glutamine, 100 µg/ml
streptomycin, and 50 µg/ml penicillin. Before use, cells were gently
scraped off with a rubber policeman and, depending on the experiment,
either plated into eight-well Labtek tissue culture chambers (Nunc,
Naperville, IL) at a concentration of 0.5 × 106
cells/well for microscopic analysis or into 12-well tissue culture dishes at a concentration of 106 cells/well in 1 ml of
culture medium (for biochemical analysis).
Yeast Culture and PLM Purification--
C. albicans
VW32 (serotype A) and S. cerevisiae Su1 (3) were used
throughout this study. Yeasts were maintained on Sabouraud's dextrose
agar (SDA) at 4 °C. Before the experiments, yeast cells were
transferred onto fresh SDA and incubated for 20 h at 37 °C. Yeast cells were then recovered, washed with phosphate-buffered saline,
and transferred into DMEM. Heat-killed yeasts were prepared by heating
20 × 106 yeasts/ml in sterile water at 95 °C for
15 min. Efficiency of killing was determined by culture of treated
yeasts on SDA for 48 h at 37 °C. The presence of PLM at the
cell wall surface of yeasts was examined by Western blot using the
anti-
PLM from C. albicans was prepared by extensive purification
partition and hydrophobic interaction steps as described previously (26). The structure of this molecule was determined by a combination of
methanolysis/HPLC, phosphorus/proton NMR, and ion spray mass spectrometry methods and is shown in Fig.
1A. This study involved the
batch of C. albicans PLM recovered from these structural
studies after analysis by nondenaturing methods.
Co-culture of Yeast Cells with Mammalian Cells and PLM
Stimulation--
J774 cells were gently scraped with a rubber
policeman and distributed into 12-well culture plates at a
concentration of 106 cells/well. After 18 h, the
adherent cells were washed with culture medium. For co-cultivation
studies, plated cells were incubated with yeasts at a concentration of
20 yeasts/J774 cell. After incubation for various times, the cultures
were washed with DMEM to remove unbound yeast cells and prepared for
either biochemical analysis or fungicidal assays. In some experiments,
cells were incubated with different concentrations of PLM in culture
medium for 1 h before the addition of yeast cells. For assessment
of the effect of PLM on macrophage responses, different concentrations
of PLM were added to plated J774 cells and incubated for various
periods of time before preparation for either biochemical analysis or fungicidal assays.
Extraction and Western Blot Analysis--
Stimulated cells were
washed with 1 ml of ice-cold phosphate-buffered saline containing 1 mM Na3VO4 and 10 mM
NaF. The cultures were extracted with 500 µl of boiling 2×
concentrated electrophoresis sample buffer (1× electrophoresis sample
buffer: 125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 1%
Extracted proteins were separated by 10% SDS-PAGE before blotting onto
a nitrocellulose membrane (Schleicher and Schuell) for 2 h at 200 mA in a semidry transfer system. After staining with 0.1% Ponceau S in
5% acetic acid to confirm equivalence of loading and transfer, the
membrane was blocked by incubation with TNT (10 mM Tris,
100 mM NaCl, 0.1% Tween) containing 5% nonfat dry milk
for 1 h at 20 °C. Membranes were probed with phosphospecific antibodies (diluted 1:1000) or anti-Bcl-2 IgG (diluted 1:250) in
TNT-5% bovine serum albumin overnight at 4 °C. After washing several times, the membranes were incubated for 1 h at 20 °C
with a 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG in TNT-5% bovine serum albumin. After washing, the membrane was
incubated with ECL detection reagents (SuperSignal Chemiluminescent Substrate) (Pierce) and exposed to hyperfilm ECL.
Immunofluorescence Analysis--
After stimulation, J774 cells
were washed with warm DMEM and fixed and permeabilized with 3.7%
formaldehyde and 0.2% Triton X-100 in phosphate buffer at 20 °C for
20 min. After three washes with phosphate buffer, 100 µl of a 1:100
dilution of anti-Bcl-2 antibody was added for 2 h at 20 °C.
After five washes, 100 µl of a 1:100 dilution of fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG in phosphate buffer was
added for 1 h at 20 °C. Slides were then washed five times and
mounted for microscopic examination.
Assessment of Apoptosis by Fluorescence
Microscopy--
Detection of apoptotic and necrotic cells after
incubation with yeasts or PLM was performed by fluorescence microscopy
with the annexin V-propidium iodide apoptosis detection kit (R&D
Systems, Minneapolis, MN) on unfixed cells as recommended by the manufacturer.
DNA Fragmentation Assay--
J774 cells (2 × 106) were collected after incubation at 37 °C for
16 h with PLM and centrifuged at 400 × g for 10 min at 20 °C. Pelleted cells were incubated for 1 h at 50 °C
in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Triton-X 100, 100 µg/ml proteinase K). The
lysates were clarified by centrifugation at 400 × g
for 30 min at 20 °C, and the supernatants were deproteinized twice
by phenol-chloroform (1:1) extraction and once by chloroform extraction. The mixtures were treated with 50% isopropyl alcohol, 0.5 M NaCl at Flow Cytometry Analysis--
Mitochondrial
alteration during apoptosis was examined in J774 cells after incubation
for 120 min without or with 10 µg/ml PLM. Treated cells were
incubated for 15 min with 10 µM YOPRO-1 (Molecular
Probes, Inc., Eugene, OR), a vital fluorescent DNA dye that allows
visualization of the alterations to the plasma membrane (30), and with
100 nM chloromethyl-X-rosamine (CMX-Ros) to reveal the
integrity of the mitochondrial transmembrane potential (31). Stained
cells were analyzed by flow cytometry (Coulter; Beckman).
Fungicidal Assays--
J774 cells were incubated for 30 min at
37 °C with yeast cells in the presence or absence of PLM, washed
with DMEM, and then recultured at 37 °C for a further 90 min. The
cultures were washed with DMEM, and endocytosed yeast cells were
released by lysing the J774 cells with sterile water. The yeast cells
recovered were counted, and 100 individual yeast cells in 1 ml of
phosphate-buffered saline were plated onto SDA. After incubation for
24 h, the number of colony-forming units was determined.
Densitometry--
Autoradiograms were scanned, and densitometry
analysis was performed using the public domain NIH Image program
(developed at the National Institutes of Health and available on the
World Wide Web at rbs.info.nih.gov/nih-image/).
C. albicans Endocytosis by J774 Cells Is Associated with
Down-regulation of Bad Ser-112 Phosphorylation--
It has previously
been shown that endocytosis of C. albicans by J774 cells is
associated with modulation of the MEK-ERK signal transduction pathway,
leading to decreased phosphorylation of ERK1/2 and its downstream
product, p90rsk (3). Phosphorylated p90rsk targets a
Ser residue at position 112 of Bad in contrast to Akt-1/protein kinase
B, which phosphorylates Bad at Ser-136 (8). As ERK1/2 and
p90rsk phosphorylation were altered after ingestion of C. albicans by J774 cells, the effect of yeast engulfment on Bad
phosphorylation was investigated. Fig. 2
shows that after incubation of J774 cells with S. cerevisiae
blastoconidia, p90rsk phosphorylation was associated with high
levels of phosphorylation of Bad at both Ser-112 and Ser-136. In cells
incubated for 15 min with C. albicans, a similar degree of
phosphorylation of p90rsk and Bad at both Ser-112 and Ser-136
was observed. However, after a 60-min incubation with C. albicans, cells displayed a dramatic decrease in phosphorylation
of p90rsk and a simultaneous and significantly lower
phosphorylation of Bad, specifically at Ser-112.
Dephosphorylation of Bad at Ser-112 by C. albicans Coincides with
the Disappearance of Bcl-2 Staining in J774 Cells--
Phosphorylation
of Bad at Ser-112 has been shown to be critical for its binding to
14-3-3 (8). The absence of such phosphorylation frees Bad, which may
complex with the antiapoptotic protein Bcl-2. The effect of endocytosis
of C. albicans on the distribution of Bcl-2 was
therefore investigated in an immunofluorescence assay. A similar
distribution of Bcl-2 was observed in untreated J774 cells (Fig.
3A) and in cells that had
ingested S. cerevisiae blastoconidia (Fig.
3B). In contrast, a strong decrease in Bcl-2 staining was observed in cells that had ingested C. albicans yeasts (Fig.
3C). Analysis by Western blotting, to detect both free and
complexed protein, revealed similar levels of Bcl-2 expression in cell
extracts (Fig. 4). Together, these data
show that the disappearance of Bcl-2 observed in the immunofluorescence
assay could not be related to down-modulation of protein expression and
suggest the formation of heterodimeric complexes between Bcl-2 and
unphosphorylated Bad.
C. albicans-induced Proapoptotic Signal in J774 Cells--
Bad
dephosphorylation, together with the disappearance of Bcl-2 staining in
cells which had ingested C. albicans, suggest that the cells
underwent apoptosis. The effect of ingestion of C. albicans (either alive or heat killed) and S. cerevisiae on the expression of phosphatidylserine at the outer
leaflet of macrophages was investigated using propidium iodine staining
to discriminate between necrosis and apoptosis. Staining with propidium
iodine was similar irrespective of how the cells had been treated,
showing that the cells were not affected by necrosis. No binding of
annexin V was observed with control cells (Fig.
5A) or with cells that had
ingested S. cerevisiae (Fig. 5B). In contrast,
binding of annexin V to cells that had ingested live C. albicans yeast cells, revealed that phosphatidylserine was exposed
at the plasma membrane of these cells (Fig. 5C). When killed
yeasts were used, no annexin V binding at the J774 cell membrane could
be observed (Fig. 5D).
PLM Induces Disregulation of the ERK1/2 Signal
Transduction Pathway--
Presence of PLM at the surface of C. albicans yeast cells is a characteristic of this pathogenic yeast,
which makes it different from other strains of Candida or
from S. cerevisiae (26, 27). Comparison of the presence
of PLM at the surface of live and heat-killed C. albicans
(Fig. 6) revealed that the molecule was
weakly detected in extracts from heat-killed yeasts compared with live
yeast cells. We thus hypothesized that PLM could play a role in the
alteration of the J774 cell response seen with C. albicans
whole live yeast cells. The effect of PLM on signal transduction was
examined by incubating cells with PLM before the addition of either
S. cerevisiae or C. albicans blastoconidia. As
shown in Fig. 7A, the addition of PLM to J774 cells led to a decreased capability of the cells to
phosphorylate ERK1/2 in response to ingestion of S. cerevisiae. This inhibition depended on the dose of PLM used, a 10 µg/ml concentration of PLM being sufficient to obtain 40% inhibition
of the signal. Incubation of J774 cells with PLM before the addition of
C. albicans also led to the extinction of ERK1/2
phosphorylation when the highest concentration of PLM was used (50 µg/ml). The influence of PLM on the ERK1/2 pathway and downstream
products was confirmed by the decreased phosphorylation of
p90rsk observed in cells pretreated with 50 µg/ml PLM before
the addition of yeasts (Fig. 7B).
PLM Induces Apoptotic Signals in J774 Cells after Ingestion of the
Sensitive Yeast S. cerevisiae and Allows Yeasts to Escape the Lytic
Activity of Cells--
The ability of PLM to alter the signaling
response induced in cells during endocytosis and its effect on the
capability of treated cells to kill endocytosed yeasts were then
investigated. As shown in Fig. 8,
treatment of J774 cells with PLM before the addition of yeasts led to a
decreased capability of J774 cells to kill S. cerevisiae.
The inhibitory effect of PLM depended on the dose used for pretreating
the cells (Fig. 8A) and was maximal with 50 µg/ml PLM.
With this dose, 28 ± 2% of blastoconidia that had been ingested
by the cells survived compared with 5 ± 5% of blastoconidia that
survived after ingestion by untreated control cells (Fig.
8B).
The effect of PLM treatment on Bcl-2 staining after ingestion of
S. cerevisiae by J774 cells was then determined (Fig.
9). As shown above (Fig. 3), cells that
had ingested S. cerevisiae presented strong Bcl-2
immunostaining (Fig. 9A). Treatment with PLM before the
addition of yeasts led to reduced, if any, staining of the protein
(Fig. 9B), which was comparable with that observed after
endocytosis of C. albicans blastoconidia (Fig. 3).
PLM Induces Apoptosis of J774 Cells through a Signal That Affects
Mitochondrial Integrity--
The direct effect of PLM on J774 cell
apoptosis was then determined. J774 cells were incubated with PLM, and
evidence of apoptosis was sought by different methods.
The effect of incubation with PLM on cell membrane permeability and
mitochondrial potential was examined by flow cytometry (Fig.
10). In the absence of PLM treatment,
86.3% of cells were negative for YOPRO-1, revealing plasma membrane
integrity of the cells (Fig. 10A), and positive by CMX-Ros
staining, revealing intact mitochondrial potential (Fig.
10B). After treatment with PLM, although most cells were
unlabeled with YOPRO-1 (Fig. 10C), more than 34% of cells
presented negative staining with CMX-Ros, showing that the mitochondria
were altered by the treatment (Fig. 10D).
The apoptotic process induced by PLM was confirmed using annexin V to
reveal phosphatidylserine expression on the external side of the
treated cell membrane. Compared with untreated cells (Fig.
11, A and C),
binding of annexin V to the cell membrane was most obvious after
incubation of the cells with PLM (Fig. 11, B and
D).
Apoptosis of cells after incubation with PLM was also confirmed by the
DNA fragmentation observed in these cells, which was related to the
dose of PLM used (Fig. 12).
Several pathogens are known to interfere with host cell apoptotic
control (32). Depending on their parasitic behavior, differential triggering of cell survival or cell death is used by
microbes to promote their survival. The yeast C. albicans has been reported to inhibit tumor necrosis
factor- In our C. albicans model, heat-killed yeasts did not induce
apoptosis. However, due to PLM hydrophylicity (conferred by its glycan
moiety), this molecule is not retained within the cell wall of dead
cells (see Fig. 6); conventional heat treatment resulted in a dramatic
decrease in the PLM associated with heat-killed yeasts placed in
contact with macrophages. This prompted us to consider PLM, rather than
any other mechanism triggered by living yeasts, as the molecule
responsible for the proapoptotic activity of C. albicans on
macrophages. The PLM proapoptotic effect was strong enough to promote
the survival of the sensitive yeast S. cerevisiae when the
C. albicans-derived molecule PLM was added together with
S. cerevisiae to macrophages (Fig. 1B).
Extensive literature exists on the effects of surface
glycolipids from pathogens on the control of host cell apoptosis (36). The lipoarabinomannan from the prokaryotic intracellular pathogen Mycobacteria tuberculosis promotes macrophage survival by
mediating phosphorylation of Bad at Ser-136 in a phosphatidylinositol
3-kinase/Akt-dependent manner (15). The lipophosphoglycan
from Leishmania donovani, which is a glycolipid present at
the cell surface of an eukaryotic intracellular pathogen belonging to
the family Trypanosomatideaea, also promotes host cell
survival (19). However, depending on the structure of the glycolipid,
opposite effects may also be observed. The glycosylinositol
phospholipid from Trypanosoma cruzi, another member
of the Trypanosomatideaea with an extracellular parasitic
phase, induces host cell apoptosis (20). However, in contrast to
lipophosphoglycan, the lipid core of glycosylinositol phospholipid belongs to a family of ceramides whose proapoptotic effects are well known (37, 38).
C. albicans PLM is expressed at the cell wall surface
of live yeasts and binds to and stimulates macrophages through a
process of shedding from the yeast cell wall initiated by contact
(26-29, 39, 40). Recent structural investigations have shown that the
PLM glycan moiety composed of long linear chains of
Ceramides have been shown to interfere either with the ERK1/2 signaling
pathway (46) or the PI-3k and Akt/protein kinase B pathway, which
regulates Bad activity at Ser-136 (47, 48). Bad dephosphorylation at
Ser-136 by prolonged inactivation of Akt/protein kinase B (49) has been
described as ceramide-induced apoptosis in Bad-expressing COS-7
cells through a mechanism involving Ras-MEK1. In this study, no
alteration of phosphorylation of Ser-136 was observed. This argues
against a role for Akt/protein kinase B in C. albicans-induced interference. This was confirmed by treatment of
cells with wortmanin, a well known inhibitor of PI-3k and consequently of Akt/protein kinase B and Bad phosphorylation at Ser-136 (50). This
treatment did not change the viability of yeasts after ingestion by
J774 cells (data not shown). The hypothesis that the PLM ceramide moiety may be responsible for apoptosis is nonetheless coherent with
the observed transduction pathways, since (i) ceramides have been shown
to accelerate dephosphorylation of ERK1/2 (46) and to inhibit cell
growth through a mechanism involving protein kinase C- In conclusion, PLM was shown to cause by itself an alteration to host
cells, which resulted in the cells undergoing apoptosis, as
revealed by mitochondrial potential alteration, phosphatidylserine exposure at the plasma membrane, and DNA fragmentation. The apoptotic pathway induced is probably of pathophysiological significance, since
killed yeasts did not display such activity and the addition of PLM
allowed S. cerevisiae to survive to macrophage
phagocytosis. The mechanisms involved in induction of host cell death
remain obscure. Although participation of other yeast factors cannot be
excluded, the unique phylogenetic structure of PLM makes it a worthy
candidate for further investigation.
-1,2-oligomannosides
and phytoceramide. This study compared the specific contribution of PLM
to the modulation of signaling pathways linked to the survival of
C. albicans in macrophages in contrast to
Saccharomyces cerevisiae. C. albicans
endocytosis by J774 and disregulation of the ERK1/2 signal transduction
pathway was associated downstream with a reduction in Bad Ser-112
phosphorylation and disappearance of free Bcl-2. This suggested an
apoptotic effect, which was confirmed by staining of phosphatidylserine
in the macrophage outer membrane. The addition of PLM to macrophages
incubated with S. cerevisiae mimicked each of the
disregulation steps observed with C. albicans and promoted
the survival of S. cerevisiae. Externalization of
membranous phosphatidylserine, loss of mitochondrial integrity, and DNA
fragmentation induced by PLM showed that this molecule promoted yeast
survival by inducing host cell death. These findings suggest strongly
that PLM is a virulence attribute of C. albicans and that
elucidation of the relationship between structure and apoptotic
activity is an innovative field of research.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6). They are
involved in maintaining the phosphorylated state of Bad, a proapoptotic
member of the Bcl-2 family that plays an important role in mediating
signal transduction pathways leading to apoptosis. Bad function is
regulated by phosphorylation at two sites, serine 112 (Ser-112) and serine 136 (Ser-136) (7). Whereas Ser-136 phosphorylation
is associated with activation of Akt, Ser-112 phosphorylation requires
activation of the mitogen-activated protein kinase pathway (8).
Phosphorylated Bad is held by the 14-3-3 protein, freeing Bcl-x(L) and
Bcl-2 to promote survival (7). Unphosphorylated Bad dissociates from
14-3-3 and recruits Bcl-2 and Bcl-x(L) to initiate events that lead to
mitochondrial dysfunction and caspase activation (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,2-oligomannoside monoclonal
antibody AF1 was provided by A. Cassone (28).
-1,2 oligomannoside monoclonal antibody AF1 as described
previously (28).
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Fig. 1.
A, structure of C. albicans
PLM determined by methanolysis/HPLC, phosphorus/proton NMR, and ion
spray mass spectrometry (n = 3-20 mannose residues,
with most PLM presenting more than 11 mannose units). B,
model of the PLM regulatory pathway showing its possible role in
induction of apoptosis. Endocytosis of yeasts is associated with
activation of the MEK-ERK1/2 pathway, leading to specific
phosphorylation of p90rsk and Bad at Ser-112 (left
panel), contrasting with Bad phosphorylation at Ser-136,
which is based on PI-3k/Akt activation (in gray). In
parallel, activation of the phosphatase, MKP-1, regulates the cell
response. S. cerevisiae endocytosis is followed by lysis of
the yeasts. With C. albicans, phosphatase is
overexpressed, leading to dephosphorylation of ERK1/2, p90rsk
and Bad at Ser-112 (right panel). This results in
apoptosis of macrophages and survival of the yeasts. The addition of
C. albicans PLM to the macrophages together with S. cerevisiae mimicked the cell alterations induced by C. albicans yeasts. This allows S. cerevisiae to survive
by promoting macrophage apoptosis. (1), results obtained in
Ref. 3.
-mercaptoethanol, and bromphenol blue). Lysates were collected and
clarified by centrifugation for 10 min at 12,000 × g
at 4 °C.
20 °C overnight for DNA precipitation.
Precipitates were pelleted, washed with 70% ethanol, air-dried, and
reconstituted with 20 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4). Aliquots (10 µl) were applied to
horizontal agarose gels (2%) and subjected to a standard
electrophoresis procedure. Gels were stained with 5 µg/ml ethidium
bromide and photographed under UV light.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Signal transduction induced after endocytosis
of yeasts by J774 cells. J774 cells were either untreated or
incubated with S. cerevisiae (S.c.) or C. albicans (C.a.) blastoconidia. After 15 or 60 min,
cells were lysed as described. Whole cell lysates were separated by
SDS-PAGE and transferred to nitrocellulose membranes. The blots were
probed with antibodies specific for phosphorylated forms of either
p90rsk, Bad Ser-112, or Bad Ser-136. Blots were developed with
ECL, and the autoradiograms were scanned. The data shown are
representative of four independent experiments.
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Fig. 3.
Staining of Bcl-2 in J774 cells after
incubation with yeasts. J774 cells were incubated for 60 min
without (A) or with either S. cerevisiae
(B) or C. albicans (C). Cells were
stained with anti-Bcl-2 antibodies and examined by fluorescence
microscopy. Data shown are the results of one experiment, which was
representative of at least five independent experiments. The
arrows indicate ingested yeasts.
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Fig. 4.
Western blotting of Bcl-2 in J774 cells after
incubation with yeasts. J774 cells were either untreated or
incubated with S. cerevisiae (S.c.) or C. albicans (C.a.) blastoconidia. After 60 min, cells were
lysed as described. Whole cell lysates were separated by SDS-PAGE and
transferred to nitrocellulose membranes. The blots were probed with
anti-Bcl-2 antibodies. Blots were developed with ECL, and the
autoradiograms were scanned. The data shown are representative of three
independent experiments.
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Fig. 5.
Exposure of phosphatidylserine after
incubation of J774 cells with yeasts. J774 cells were incubated
for 90 min without (A) or with either S. cerevisiae (B), C. albicans live yeasts
(C), or heat-killed C. albicans (D).
Detection was performed by fluorescence microscopy with
fluorescein isothiocyanate-conjugated annexin V in the presence
of propidium iodide. Data shown are representative of four independent
experiments.
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Fig. 6.
Expression of PLM at the surface of
C. albicans yeast cells. After culture
on SDA for 20 h at 37 °C, yeasts were killed by heating 15 min
at 95 °C. Surface extracts from different amounts of yeast cells
either alive or heat-killed were separated by 7-20% gradient SDS-PAGE
and transferred to nitrocellulose membranes. The presence of PLM was
detected by probing the blots with anti- -1,2-oligomannoside
monoclonal antibody and developed by ECL (A). The results
presented in the histogram (B) are the mean band intensity
in arbitrary units as quantified by densitometry analysis. The data
shown are representative of three independent experiments.
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Fig. 7.
Modulation of ERK1/2 and p90rsk
phosphorylation by PLM. J774 cells were incubated with different
concentrations (0 50 µg/ml) of PLM for 60 min at 37 °C. Either
S. cerevisiae or C. albicans blastoconidia were
then added or not (none) to the cells. After a 60-min
incubation, the cells were washed and extracted. Whole cell lysates
were separated by SDS-PAGE and transferred to nitrocellulose membranes.
The blots were probed with antibodies specific for phosphorylated forms
of ERK1/2 (A) or p90rsk (B). Blots were
developed with ECL, and the autoradiograms were scanned. The data shown
are representative of four independent experiments.
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Fig. 8.
Effect of PLM treatment on yeast survival
after phagocytosis. J774 cells were untreated or pretreated with
different concentrations of PLM for 60 min at 37 °C. S. cerevisiae blastoconidia were then added (at a 1:20 cell/yeast
ratio) and cultured with the cells for 30 min. Free yeasts were
discarded, and endocytosed yeasts were recovered after 90 min by lysis
of J774 cells. 100 yeasts were transferred onto SDA. The number of
colony-forming units (cfu) was scored after 24 h.
A, results of one experiment showing the
dose-dependent effect of pretreatment with PLM.
B, results are expressed as the mean ± S.D. of four
independent experiments.
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Fig. 9.
Effect of treatment of J774 cells with PLM on
Bcl-2 staining after ingestion of S. cerevisiae.
J774 cells were left untreated (A) or treated for 60 min
with 50 µg/ml PLM (B) before incubation for 60 min with
S. cerevisiae blastoconidia. After washing, cells were
stained with anti-Bcl-2 antibodies and examined by fluorescence
microscopy. Data shown are the results of one experiment, which was
representative of at least five independent experiments.
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Fig. 10.
Cytometry analysis of plasma membrane and
mitochondrial integrity of J774 cells after incubation with PLM.
J774 cells were either left untreated (A and B)
or treated with PLM (C and D) for 120 min at
37 °C. After washing, the cells were recovered by trypsin treatment
and incubated for 15 min with 10 µM YOPRO-1 (A
and C) to examine plasma membrane alterations and 100 nM CMX-Ros (B and D) to demonstrate
the integrity of the mitochondrial transmembrane potential. Staining
was analyzed by flow cytometry.
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Fig. 11.
Phosphatidylserine exposure after incubation
of J774 cells with PLM. J774 cells were incubated for 60 min
without (A and C) or with 50 µg/ml PLM
(B and D). Detection was performed by
fluorescence microscopy with fluorescein isothiocyanate-conjugated
annexin V in the presence of propidium iodide. A and
B, double staining with annexin V and propidium iodide.
A and B, annexin V staining alone. C
and D, double staining with annexin V and propidium iodide.
Data shown are representative of four independent experiments.
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[in a new window]
Fig. 12.
DNA fragmentation of J774 cells induced by
PLM. J774 cells were incubated at 37 °C for 16 h with
different concentrations of PLM. Cells were collected, and DNA was
extracted. 10 µl of purified DNA was applied to horizontal agarose
gels (2%) and subjected to electrophoresis. Gels were stained with
ethidium bromide and photographed under UV light. DNA size markers are
shown on the left. Data shown are representative of three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced DNA fragmentation in macrophages (33) and to induce
apoptosis of macrophages (24) and neutrophils (25), but the mechanisms
and C. albicans molecules involved are unknown. It has
previously been shown that, in contrast to the nonpathogenic yeast
S. cerevisiae, C. albicans was able to
specifically alter signal transduction of the MEK-ERK pathway (3). This
down-modulation of the cell machinery decreased phosphorylation of
p90rsk, resulting in survival of ingested yeast cells. In this
study, the alteration of ERK1/2 and p90rsk phosphorylation was
observed to end with the specific dephosphorylation of Bad at Ser-112.
Dephosphorylation of Bad, which allows it to complex with
anti-apoptotic Bcl-2 homologues, is known to induce mitochondrial
events leading to apoptosis (9, 35).
-1,2-oligomannosides (40) coupled by a specific arm to
phytoceramide, is derived from the mannose inositol
phosphoceramide biosynthetic pathway (26). Although
-1,2-oligomannosides per se have been demonstrated to act
as adhesins (41, 42) and stimulate macrophages (43, 44), the
proapoptotic properties of PLM are more likely to be related to its
lipid core (38, 45).
(51) and (ii)
the down-regulation of ERK1/2 previously observed after endocytosis of
C. albicans was related to prolonged activation of MKP-1, a
MEK-ERK-specific phosphatase whose expression also depends on
n-protein kinase C activation (3). However, the mechanisms by which
ceramide may induce apoptosis are multiple. It has recently been shown
that ceramide may induce mitochondrial activation through Bax, a
proapoptotic member of the Bcl-2 family closely related to Bad. This
apoptosis was independent of caspase activation (52). Whether
PLM-induced apoptosis depends on caspase activation remains to be
determined. Endocytosis of Escherichia coli by macrophages
has recently been described to activate the proapoptotic signal (53).
In this case, signaling was initiated by Toll-like receptor-2 and
involved the adaptor, myeloid differentiation factor 88, and the
caspase-9/-3-dependent mitochondrial amplification loop.
Myeloid differentiation factor 88 may also directly recruit caspase-8
(54). Interestingly, both the mitochondrial amplification loop and
caspase-8 have recently been shown to be modulated by ERK
phosphorylation (55). These differential mechanisms by which ceramide
influences the balance between cell survival and cell death are
probably closely related to their structure. In this respect, the
phytoceramide nature of the C. albicans PLM lipid moiety
raises an important question (26). Although our knowledge of
phytoceramide and phytosphingosine synthesis or hydrolysis is still
embryonic, the presence of these molecules in mammalian cells has been
demonstrated, and several studies have suggested that, like ceramide,
phytosphingosine may play an important role in cell growth or death of
eukaryotic cells (56, 57). Due to their critical role in cell
integrity, it is interesting to note that ceramide biosynthetic
pathways are also critical for the establishment of fungal pathogens in
the host. For the intracellular pathogen Cryptococcus
neoformans, it has been recently shown that down-regulation of
C. neoformans inositolphosphoryl phytoceramide synthase (an
early step in mannose inositol phosphoceramide synthesis) significantly decreased intracellular growth and resistance of this
yeast in J774 macrophages (34).
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ACKNOWLEDGEMENT |
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We gratefully acknowledge the technical assistance of Nathalie Jouy (Inserm IFR114).
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FOOTNOTES |
---|
* This work was supported by the "Réseau Infection Fongique" of the French Ministère de l'Education Nationale, de la Recherche et de la Technologie (MENRT).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ S. Ibata-Ombetta was supported by a grant from the Conseil Régional du Nord-Pas de Calais.
To whom correspondence should be addressed: Laboratoire de
Mycologie Fondamentale et Appliquée, Université de Lille
II, Faculté de Médecine H. Warembourg, Pôle
Recherche, Place Verdun, 59037 Lille Cedex, France. Tel.:
33-3-20-62-34-15; Fax: 33-3-20-62-34-16; E-mail:
tjouault@univ-lille2.fr.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210680200
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ABBREVIATIONS |
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The abbreviations used are: PLM, phospholipomannan; ERK, extracellular signal-regulated kinase; SDA, Sabouraud's dextrose agar; DMEM, Dulbecco's modified Eagle's medium; HPLC, high pressure liquid chromatography; CMX-Ros, chloromethyl-X-rosamine; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.
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REFERENCES |
---|
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---|
1. | Pfaller, M. A., Jones, R. N., Messer, S. A., Edmond, M. B., and Wenzel, R. P. (1998) Diagn. Microbiol. Infect. Dis. 30, 121-129[CrossRef][Medline] [Order article via Infotrieve] |
2. | Calderone, R. A., and Fonzi, W. A. (2001) Trends Microbiol. 9, 327-335[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Ibata-Ombetta, S.,
Jouault, T.,
Trinel, P. A.,
and Poulain, D.
(2001)
J. Leukocyte Biol.
70,
149-154 |
4. | Jin, K., Mao, X. O., Zhu, Y., and Greenberg, D. A. (2002) J. Neurochem. 80, 119-125[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Tan, Y.,
Ruan, H.,
Demeter, M. R.,
and Comb, M. J.
(1999)
J. Biol. Chem.
274,
34859-34867 |
6. |
Tran, S. E.,
Holmstrom, T. H.,
Ahonen, M.,
Kahari, V. M.,
and Eriksson, J. E.
(2001)
J. Biol. Chem.
276,
16484-16490 |
7. |
Masters, S. C.,
and Fu, H.
(2001)
J. Biol. Chem.
276,
45193-45200 |
8. |
Scheid, M. P.,
Schubert, K. M.,
and Duronio, V.
(1999)
J. Biol. Chem.
274,
31108-31113 |
9. | Korsmeyer, S. J. (1999) Cancer Res. 59, 1693-1700 |
10. | Navarre, W. W., and Zychlinsky, A. (2000) Cell Microbiol. 2, 265-273[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Santos, R. L.,
Tsolis, R. M.,
Baumler, A. J.,
Smith, R.,
and Adams, L. G.
(2001)
Infect Immun.
69,
2293-2301 |
12. |
Shibayama, K.,
Doi, Y.,
Shibata, N.,
Yagi, T.,
Nada, T.,
Iinuma, Y.,
and Arakawa, Y.
(2001)
Infect Immun.
69,
3181-3189 |
13. | Kornfeld, H., Mancino, G., and Colizzi, V. (1999) Cell Death Differ. 6, 71-78[CrossRef][Medline] [Order article via Infotrieve] |
14. | Heussler, V. T., Kuenzi, P., and Rottenberg, S. (2001) Int. J. Parasitol. 31, 1166-1176[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Maiti, D.,
Bhattacharyya, A.,
and Basu, J.
(2001)
J. Biol. Chem.
276,
329-333 |
16. |
Aga, E.,
Katschinski, D.,
van Zandbergen, G.,
Laufs, H.,
Hansen, B.,
Muller, K.,
Solbach, W.,
and Laskay, T.
(2002)
J. Immunol.
169,
898-905 |
17. | Luder, C. G., Gross, U., and Lopes, M. F. (2001) Trends Parasitol. 17, 480-486[CrossRef][Medline] [Order article via Infotrieve] |
18. | Gao, L. Y., and Kwaik, Y. A. (2000) Trends Microbiol. 8, 306-313[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Moore, K. J.,
and Matlashewski, G.
(1994)
J. Immunol.
152,
2930-2937 |
20. |
Freire-de-Lima, C. G.,
Nunes, M. P.,
Corte-Real, S.,
Soares, M. P.,
Previato, J. O.,
Mendonca-Previato, L.,
and DosReis, G. A.
(1998)
J. Immunol.
161,
4909-4916 |
21. |
Cornelis, G. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8778-8783 |
22. | Orth, K. (2002) Curr. Opin. Microbiol. 5, 38-43[CrossRef][Medline] [Order article via Infotrieve] |
23. | Calderone, R. (2002) Candida and Candidiasis , ASM Press, Washington, D. C. |
24. | Schroppel, K., Kryk, M., Herrmann, M., Leberer, E., Rollinghoff, M., and Bogdan, C. (2001) Int. J. Med. Microbiol. 290, 659-668[Medline] [Order article via Infotrieve] |
25. | Rotstein, D., Parodo, J., Taneja, R., and Marshall, J. C. (2000) Shock 14, 278-283[Medline] [Order article via Infotrieve] |
26. |
Trinel, P. A.,
Maes, E.,
Zanetta, J. P.,
Delplace, F.,
Coddeville, B.,
Jouault, T.,
Strecker, G.,
and Poulain, D.
(2002)
J. Biol. Chem.
277,
37260-37271 |
27. |
Poulain, D.,
Slomianny, C.,
Jouault, T.,
Gomez, J.,
and Trinel, P.
(2002)
Infect. Immun.
70,
4323-4328 |
28. | Jouault, T., Fradin, C., Trinel, P. A., Bernigaud, A., and Poulain, D. (1998) J Infect. Dis. 178, 792-802[Medline] [Order article via Infotrieve] |
29. | Jouault, T., Bernigaud, A., Lepage, G., Trinel, P. A., and Poulain, D. (1994) Immunology 83, 268-273[Medline] [Order article via Infotrieve] |
30. | Idziorek, T., Estaquier, J., De Bels, F., and Ameisen, J. C. (1995) J. Immunol. Methods 185, 249-258[CrossRef][Medline] [Order article via Infotrieve] |
31. | Gilmore, K., and Wilson, M. (1999) Cytometry 36, 355-358[CrossRef][Medline] [Order article via Infotrieve] |
32. | Monack, D., and Falkow, S. (2000) Int. J. Med. Microbiol. 290, 7-13[Medline] [Order article via Infotrieve] |
33. | Heidenreich, S., Otte, B., Lang, D., and Schmidt, M. (1996) J. Leukocyte Biol. 60, 737-743[Abstract] |
34. |
Luberto, C.,
Toffaletti, D. L.,
Wills, E. A.,
Tucker, S. C.,
Casadevall, A.,
Perfect, J. R.,
Hannun, Y. A.,
and Del Poeta, M. M.
(2001)
Genes Dev.
15,
201-212 |
35. |
Gross, A.,
McDonnell, J. M.,
and Korsmeyer, S. J.
(1999)
Genes Dev.
13,
1899-1911 |
36. | Ropert, C., and Gazzinelli, R. T. (2000) Curr. Opin. Microbiol. 3, 395-403[CrossRef][Medline] [Order article via Infotrieve] |
37. | Rodriguez-Lafrasse, C., Alphonse, G., Broquet, P., Aloy, M. T., Louisot, P., and Rousson, R. (2001) Biochem. J. 357, 407-416[CrossRef][Medline] [Order article via Infotrieve] |
38. | Liu, G., Kleine, L., and Hebert, R. L. (1999) Crit. Rev. Clin. Lab. Sci. 36, 511-573[Medline] [Order article via Infotrieve] |
39. | Trinel, P. A., Borg-von-Zepelin, M., Lepage, G., Jouault, T., Mackenzie, D., and Poulain, D. (1993) Infect. Immun. 61, 4398-4405[Abstract] |
40. |
Trinel, P. A.,
Plancke, Y.,
Gerold, P.,
Jouault, T.,
Delplace, F.,
Schwarz, R. T.,
Strecker, G.,
and Poulain, D.
(1999)
J. Biol. Chem.
274,
30520-30526 |
41. | Fradin, C., Jouault, T., Mallet, A., Mallet, J. M., Camus, D., Sinay, P., and Poulain, D. (1996) J. Leukocyte Biol. 60, 81-87[Abstract] |
42. |
Fradin, C.,
Poulain, D.,
and Jouault, T.
(2000)
Infect. Immun.
68,
4391-4398 |
43. |
Jouault, T.,
Fradin, C.,
Trinel, P. A.,
and Poulain, D.
(2000)
Infect. Immun.
68,
965-968 |
44. | Jouault, T., Lepage, G., Bernigaud, A., Trinel, P. A., Fradin, C., Wieruszeski, J. M., Strecker, G., and Poulain, D. (1995) Infect. Immun. 63, 2378-2381[Abstract] |
45. | Ruvolo, P. P. (2001) Leukemia 15, 1153-1160[CrossRef][Medline] [Order article via Infotrieve] |
46. | Kitatani, K., Akiba, S., Hayama, M., and Sato, T. (2001) Arch. Biochem. Biophys. 395, 208-214[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Kanto, T.,
Kalinski, P.,
Hunter, O. C.,
Lotze, M. T.,
and Amoscato, A. A.
(2001)
J. Immunol.
167,
3773-3784 |
48. | Kim, D. S., Kim, S. Y., Moon, S. J., Chung, J. H., Kim, K. H., Cho, K. H., and Park, K. C. (2001) Pigment Cell Res. 14, 110-115[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Basu, S.,
Bayoumy, S.,
Zhang, Y.,
Lozano, J.,
and Kolesnick, R.
(1998)
J. Biol. Chem.
273,
30419-30426 |
50. |
Cantley, L. C.
(2002)
Science
296,
1655-1657 |
51. | Bourbon, N. A., Yun, J., Berkey, D., Wang, Y., and Kester, M. (2001) Am. J. Physiol. 280, C1403-C1411 |
52. | von Haefen, C., Wieder, T., Gillissen, B., Starck, L., Graupner, V., Dorken, B., and Daniel, P. T. (2002) Oncogene 21, 4009-4019[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Hacker, H.,
Furmann, C.,
Wagner, H.,
and Hacker, G.
(2002)
J. Immunol.
169,
3172-3179 |
54. |
Aliprantis, A. O.,
Yang, R. B.,
Weiss, D. S.,
Godowski, P.,
and Zychlinsky, A.
(2000)
EMBO J.
19,
3325-3336 |
55. |
Soderstrom, T.,
Poukkula, M.,
Holmstrom, T.,
Heiskanen, K.,
and Eriksson, J.
(2002)
J. Immunol.
169,
2851-2860 |
56. | Tamiya-Koizumi, K., Murate, T., Suzuki, M., Simbulan, C. M., Nakagawa, M., Takemura, M., Furuta, K., Izuta, S., and Yoshida, S. (1997) Biochem. Mol. Biol. Int. 41, 1179-1189[Medline] [Order article via Infotrieve] |
57. | Lee, J. S., Min, D. S., Park, C., Park, C. S., and Cho, N. J. (2001) FEBS Lett. 499, 82-86[CrossRef][Medline] [Order article via Infotrieve] |