Candida albicans phospholipomannan promotes survival of ... · 1/27/2003 · Stella...

Candida Phospholipomannan-induced apoptosis

1

Candida albicans phospholipomannan promotes survival of phagocytozed yeasts through

modulation of Bad phosphorylation and macrophage apoptosis

Stella Ibata-Ombetta1, Thierry Idziorek2, Pierre-André Trinel1,

Daniel Poulain1 and Thierry Jouault1*

1Laboratoire de Mycologie Fondamentale et Appliquée, Inserm EMI0360, Université de Lille

II, and 2Inserm U459, Faculté de Médecine H. Warembourg, Place Verdun, 59037 Lille

Cedex, France

*Corresponding author: Thierry Jouault, 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: [email protected]

Running title: Candida phospholipomannan-induced apoptosis

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 27, 2003 as Manuscript M210680200 by guest on O

ctober 30, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


2

SUMMARY

The surface of the pathogenic yeast Candida albicans is coated with phospholipomannan

(PLM), a phylogenetically unique glycolipid composed of b-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 to the survival of

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.

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.

by guest on October 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


3

INTRODUCTION

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 underway 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

non-pathogenic 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 (4-6). They are involved in maintaining the phosphorylated state of

Bad, a pro-apoptotic 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). While Ser-

136 phosphorylation is associated with activation of Akt, Ser-112 phosphorylation requires

activation of the MAPK 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).

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


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


4

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), composed of

oligomannose residues with a unusual type of linkage and phytoceramide (26,27) (Figure 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. 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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


5

EXPERIMENTAL PROCEDURES

Reagents and antibodies

All reagents were obtained from Sigma (Sigma Aldrich Chimie, Saint Quentin

Fallavier, France). Specific rabbit polyclonal IgGs to the phosphorylated forms of Bad (Ser-

112 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 (Santa

Cruz, CA). Horseradish peroxidase (HRPO) and FITC-conjugated anti-rabbit IgG were

obtained from Southern Biotechnology Laboratories (Birmingham, AL). Anti-b-1,2

oligomannosides monoclonal antibody AF1 was provided by A. Cassone (28).

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 8-well Labtek tissue

culture chambers (Nunc, Naperville, IL) at a concentration of 0.5 x 106 cells per well for

microscopic analysis, or into 12-well tissue culture dishes at a concentration of 106 cells per

well in 1 ml of culture medium (for biochemical analysis).

Yeasts 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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


6

Yeast cells were then recovered, washed with phosphate-buffered saline (PBS) and

transferred into DMEM. Heat killed yeasts were prepared by heating 20 x 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. Presence of PLM at the cell wall surface of yeasts was

examined by western blot using the anti-b-1,2 oligomannoside mAb AF1 as described

previously (28).

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 Figure 1A. This study involved the batch of C.

albicans PLM recovered from these structural studies after analysis by non-denaturing

methods.

Figure 1

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 per 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 per 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 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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


7

Extraction and Western blot analysis

Stimulated cells were washed with 1 ml of ice cold PBS containing 1 mM Na3VO4 and

10 mM NaF. The cultures were extracted with 500 µl boiling 2 x concentrated electrophoresis

sample buffer (1 x : 125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 1% b-mercaptoethanol

and bromophenol blue). Lysates were collected and clarified by centrifugation for 10 min at

12 000 x g at 4°C.

Extracted proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide

gel electrophoresis (SDS-PAGE) before blotting onto a nitrocellulose membrane (Schleicher

and Schuell, Dassel, Germany) for 2 h at 200 mA in a semi-dry 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 (Tris 10 mM, NaCl 100 mM, Tween 0.1%)

containing 5% non-fat dry milk for 1 h at 20°C. Membranes were probed with phospho-

specific antibodies (diluted 1:1000) or anti-Bcl-2 IgG (diluted 1:250) in TNT-5% bovine

serum albumin (BSA) overnight at 4°C. After washing several times, the membranes were

incubated for 1 h at 20°C with a 1:2000 dilution of HRPO-conjugated anti-rabbit IgG in TNT-

5% BSA. After washing, the membrane was incubated with ECL detection reagents

(SuperSignal Chemiluminescent Substrate; Pierce, Rockford, IL, USA) 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 FITC-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


8

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, USA) on unfixed cells as recommended by

the manufacturer.

DNA fragmentation assay

J774 cells (2 x 106) were collected after incubation at 37°C for 16 h with PLM, and

centrifuged at 400 x 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 x g for 30 min at 20°C and

the supernatants were de-proteinized twice by phenol-chloroform (1:1) extraction and once by

chloroform extraction. The mixtures were treated with 50% isopropanol/0.5 M NaCl at -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.

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


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


9

µM YOPRO-1 (Molecular Probes, Eugene, OR, USA), a vital fluorescent DNA dye that

allows visualisation 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 endocytozed 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 PBS 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 NIH and available on the Internet at:

http://rbs.info.nih.gov/nih-image/ ).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


10

RESULTS

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/PKB,

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. Figure 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 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.

Figure 2

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 anti-

apoptotic 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


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


11

was observed in untreated J774 cells (Figure 3A) and in cells which had ingested S. cerevisiae

blastoconidia (Figure 3B). In contrast, a strong decrease in Bcl-2 staining was observed in

cells that had ingested C. albicans yeasts (Figure 3C). Analysis by western blotting, to detect

both free and complexed protein, revealed similar levels of Bcl-2 expression in cell extracts

(Figure 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.

Figure 3, Figure 4

C. albicans-induced pro-apoptotic 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 (Figure

5A) or with cells that had ingested S. cerevisiae (Figure 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 (Figure 5C). When killed yeasts were used, no

annexin V binding at the J774 cells membrane could be observed (Figure 5D).

Figure 5


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


12

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. cerevisae

(26, 27). Comparison of the presence of PLM at the surface of live and heat killed C. albicans

(Figure 6) revealed that the molecule was weakly detected in extracts from heat killed yeasts

compared to 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 addition of

either S. cerevisiae or C. albicans blastoconidia. As shown in Figure 7A, 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 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 pre-treated with 50 µg/ml of PLM before addition

of yeasts (Figure 7B).

Figure 6, 7

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 endocytozed yeasts were then

investigated. As shown in Figure 8, treatment of J774 cells with PLM before addition of


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


13

yeasts led to a decreased capability of J774 cells to kill S. cerevisiae. The inhibitory effect of

PLM depended on the dose used for pre-treating the cells (Figure 8A) and was maximal with

50 µg/ml of PLM. With this dose, 28 ± 2% of blastoconidia that had been ingested by the

cells survived compared with 5 ± 5% of blastoconidia which survived after ingestion by

untreated control cells (Figure 8B).

Figure 8

The effect of PLM treatment on Bcl-2 staining after ingestion of S. cerevisiae by J774

cells was then determined (Figure 9). As shown above (Figure 3), cells that had ingested S.

cerevisiae presented strong Bcl-2 immunostaining (Figure 9A). Treatment with PLM before

addition of yeasts led to reduced, if any, staining of the protein (Figure 9B), which was

comparable to that observed after endocytosis of C. albicans blastoconidia (Figure 3).

Figure 9

PLM induced 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 (Figure 10). In the absence of PLM treatment,

86.3% of cells were negative for YOPRO-1, revealing plasma membrane integrity of the cells

(Figure 10A), and positive by CMX-Ros staining, revealing intact mitochondrial potential

(Figure 10B). After treatment with PLM, although most cells were unlabelled with YOPRO-1

(Figure 10C), more than 34% of cells presented negative staining with CMX-Ros showing

that the mitochondria were altered by the treatment (Figure 10D).

Figure 10


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


14

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 to

untreated cells (Figure 11A, C), binding of annexin V to the cell membrane was most obvious

after incubation of the cells with PLM (Figure 11B, 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 (Figure 12).

Figure 11, Figure 12


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


15

DISCUSSION

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

TNFa-induced DNA fragmentation in macrophages (33) and to induce apoptosis of

macrophages (34) and neutrophils (25), but the mechanisms and C. albicans molecules

involved are unknown. It has previously been shown that, in contrast to the non-pathogenic

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).

In our C. albicans model, heat-killed yeasts did not induce apoptosis. However, due to

PLM hydrophylicity (confered by its glycan moeity) this molecule is not retained within the

cell wall of dead cells (see Figure 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 pro-apoptotic activity of C. albicans on

macrophages. The PLM Pro-apoptotic 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 (Figure 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


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


16

phosphorylation of Bad at Ser-136 in a PI-3k/Akt-dependent manner (15). The

lipophosphoglycan (LPG) 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 glycosylinositolphospholipid (GIPL) from

Trypanosoma cruzi, another member of the Trypanosomatideaea with an extracellular

parasitic phase, induces host cell apoptosis (20). However, in contrast to LPG, the lipid core

of GIPL belongs to a family of ceramides whose pro-apoptotic 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 b-1,2 oligomannosides (40) coupled by a specific

arm to phytoceramide, is derived from the mannosylinositolphosphoryceramide (MIPC)

biosynthetic pathway (26). Although b-1,2 oligomannosides per se have been demonstrated to

act as adhesins (41,42) and stimulate macrophages (43,44), the pro-apoptotic properties of

PLM are more likely to be related to its lipid core (38,45).

Ceramides have been shown to interfere either with the ERK1/2 signaling pathway

(46) or the PI-3k and Akt/PKB pathway, which regulates Bad activity at Ser-136 (47,48). Bad

dephosphorylation at Ser-136 by prolonged inactivation of Akt/PKB (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/PKB in C. albicans-induced interference. This

was confirmed by treatment of cells with wortmanin, a well-known inhibitor of PI-3k and


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


17

consequently of Akt/PKB 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

PKCe (51); and (ii) the down-regulation of ERK1/2 previously observed after endocytosis of

C. albicans was related to prolonged activation of MKP-1, an MEK-ERK specific

phosphatase whose expression also depends on nPKC 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 pro-apoptotic

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 pro-apoptotic signal (53). In this case, signaling was initiated by Toll-like receptor

(TLR)-2 and involved the adaptor, MyD88, and the caspase-9/-3-dependent mitochondrial

amplification loop. MyD88 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


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


18

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 MIPC synthesis) significantly

decreased intracellular growth and resistance of this yeast in J774 macrophages (58).

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 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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


19

REFERENCES

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.

2. Calderone, R. A., and Fonzi, W. A. (2001) Trends Microbiol 9, 327-335.

3. Ibata-Ombetta, S., Jouault, T., Trinel, P. A., and Poulain, D. (2001) J Leukoc Biol 70,

149-154.

4. Jin, K., Mao, X. O., Zhu, Y., and Greenberg, D. A. (2002) J Neurochem 80, 119-125.

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, 1693s-1700s.

10. Navarre, W. W., and Zychlinsky, A. (2000) Cell Microbiol 2, 265-273.

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.

14. Heussler, V. T., Kuenzi, P., and Rottenberg, S. (2001) Int J Parasitol 31, 1166-1176.

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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


20

17. Luder, C. G., Gross, U., and Lopes, M. F. (2001) Trends Parasitol 17, 480-486.

18. Gao, L. Y., and Kwaik, Y. A. (2000) Trends Microbiol 8, 306-313.

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.

23. Calderone, R. (2002) Candida and Candidiasis, ASM Press, Washington DC.

24. Schroppel, K., Kryk, M., Herrmann, M., Leberer, E., Rollinghoff, M., and Bogdan, C.

(2001) Int J Med Microbiol 290, 659-668.

25. Rotstein, D., Parodo, J., Taneja, R., and Marshall, J. C. (2000) Shock 14, 278-283.

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.

29. Jouault, T., Bernigaud, A., Lepage, G., Trinel, P. A., and Poulain, D. (1994) Immunology

83, 268-273.

30. Idziorek, T., Estaquier, J., De Bels, F., and Ameisen, J. C. (1995) J Immunol Methods

185, 249-258.

31. Gilmore, K., and Wilson, M. (1999) Cytometry 36, 355-358.

32. Monack, D., and Falkow, S. (2000) Int J Med Microbiol 290, 7-13.

33. Heidenreich, S., Otte, B., Lang, D., and Schmidt, M. (1996) J Leukoc Biol 60, 737-743.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


21

34. Schroppel, K., Kryk, M., Herrmann, M., Leberer, E., Rollinghoff, M., and Bogdan, C.

(2001) Int J Med Microbiol 290, 659-668.

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.

37. Rodriguez-Lafrasse, C., Alphonse, G., Broquet, P., Aloy, M. T., Louisot, P., and

Rousson, R. (2001) Biochem J 357, 407-416.

38. Liu, G., Kleine, L., and Hebert, R. L. (1999) Crit Rev Clin Lab Sci 36, 511-573.

39. Trinel, P. A., Borg-von-Zepelin, M., Lepage, G., Jouault, T., Mackenzie, D., and Poulain,

D. (1993) Infect Immun 61, 4398-4405.

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 Leukoc Biol 60, 81-87.

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.

45. Ruvolo, P. P. (2001) Leukemia 15, 1153-1160.

46. Kitatani, K., Akiba, S., Hayama, M., and Sato, T. (2001) Arch Biochem Biophys 395,

208-214.

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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


22

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 Cell

Physiol 280, C1403-1411.

52. von Haefen, C., Wieder, T., Gillissen, B., Starck, L., Graupner, V., Dorken, B., and

Daniel, P. T. (2002) Oncogene 21, 4009-4019.

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.

57. Lee, J. S., Min, D. S., Park, C., Park, C. S., and Cho, N. J. (2001) FEBS Lett 499, 82-86.

58. 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.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


23

ABBREVIATIONS: ERK, extracellular signal-regulated kinase; GIPL, glucose-inositol

phospholipid; MIPC, mannose inositol phosphoceramide; MyD88, myeloid differentiation

factor 88; PLM, phospholipomannan; SDA, Sabouraud's dextrose agar; TLR, Toll-like

receptor

ACKNOWLEDGMENTS

We gratefully acknowledge the help of Nathalie Jouy (Inserm IFR114) for technical

assistance.

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). S. Ibata-Ombetta was

supported by a grant from the Conseil Régional du Nord-Pas de Calais.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


24

FIGURE LEGENDS

Figure 1. ( A ) Structure of C. albicans phospholipomannan (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 grey). 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 over-expressed leading to

dephosphorylation of ERK1/2, p90rsk and Bad at Ser-112 (right panel). This resultes in

apoptosis of macrophages and survival of the yeasts. 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) refers to

the results obtained in ref. (3)

Figure 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 lyzed 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 scanned. The data shown are representative of

four independent experiments.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


25

Figure 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.

Figure 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 lyzed 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 scanned. The data shown

are representative of 3 independent experiments.

Figure 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

FITC-conjugated annexin V in the presence of propidium iodide. Data shown are

representative of four independent experiments.

Figure 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. Presence of PLM was detected by

probing the blots with anti-b-1,2 oligomannoside mAb and developed by ECL (A). The

results presented in the histogram (B) are the mean band intensity in arbitrary units as


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


26

quantified by densitometry analysis. The data shown are representative of three independent

experiments.

Figure 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 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 scanned. The data shown are representative of

four independent experiments.

Figure 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 endocytozed yeasts were recovered after 90 min by lysis

of J774 cells. One-hundred 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 pre-treatment with PLM. (B) Results are expressed as the mean ± SD of four

independent experiments.

Figure 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 of 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


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


27

the results of one experiment, which was representative of at least five independent

experiments.

Figure 10. Cytometry analysis of plasma membrane and mitochondrial integrity of J774 cells

after incubation with PLM. J774 cells were either left untreated (A, B) or treated with PLM

(C, 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, C) to examine plasma membrane alterations,

and 100 nM CMX-Ros (B, D) to demonstrate the integrity of the mitochondrial

transmembrane potential. Staining was analyzed by flow cytometry.

Figure 11. Phosphatidylserine exposure after incubation of J774 cells with PLM. J774 cells

were incubated for 60 min without (A, C) or with 50 µg/ml of PLM (B, D). Detection was

performed by fluorescence microscopy with FITC-conjugated annexin V in the presence of

propidium iodide. (A, B) double staining with annexin V and propidium iodide. (C, D)

annexin V staining alone. Data shown are representative of four independent experiments.

Figure 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. Ten 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 hand side. Data shown are representative of

three independent experiments.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


28

FIGURE 1


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


29

FIGURE 2


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


30

FIGURE 3


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


31

FIGURE 4


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


32

FIGURE 5


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


33

FIGURE 6

0

10

20

30

40

50

0.5 1 5

Yeast cells (x106 )

B


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


34

FIGURE 7


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


35

FIGURE 8


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


36

FIGURE 9


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


37

FIGURE 10


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


38

FIGURE 11


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


39

FIGURE 12


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

JouaultStella Ibata-Ombetta, Thierry Idziorek, Pierre-André Trinel, Daniel Poulain and Thierry

through modulation of Bad phosphorylation and macrophage apoptosisCandida albicans phospholipomannan promotes survival of phagocytozed yeasts

published online January 27, 2003J. Biol. Chem.

10.1074/jbc.M210680200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M210680200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M210680200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/01/27/jbc.M210680200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/01/27/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/01/27/jbc.M210680200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

Candida albicans phospholipomannan promotes survival of ... · 1/27/2003 · Stella...

Documents

Transcript of Candida albicans phospholipomannan promotes survival of ... · 1/27/2003 · Stella...