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Candida albicans Phospholipomannan Promotes Survival of Phagocytosed Yeasts

Candida albicans Phospholipomannan Promotes Survival of

Phagocytosed Yeasts

Stella Ibata-Ombetta, Thierry Idziorek¶, Pierre-Andre´ Trinel, Daniel Poulain,

and Thierry Jouault

"The surface of the pathogenic yeast Candida albicans is coated with phospholipomannan (PLM), a phylogenetically unique glycolipid composed of _-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. 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.

The yeast C. albicans has been reported to inhibit tumor necrosis factor-_-induced DNA fragmentation in macrophages (33) and to induce apoptosis of macrophages (24) and neutrophils.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. Extensive literature exists on the effects of surface glycolipids from pathogens on the control of host cell apoptosis."

Candida albicans is part of the normal microbial flora that
colonizes the mucocutaneous surfaces of the oral cavity, gas-
trointestinal 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). Endo-
cytosis of C. albicans by macrophages was specifically associ-
ated 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
proapoptotic member of the Bcl-2 family that plays an impor-
tant 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 activa-
tion of Akt, Ser-112 phosphorylation requires activation of
the mitogen-activated protein kinase pathway (8). Phospho-
rylated Bad is held by the 14-3-3 protein, freeing Bcl-x(L) and
Bcl-2 to promote survival (7). Unphosphorylated Bad disso-
ciates 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 sur-
vival (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). Macro-
phages undergo apoptotic cell death after infection with
C. albicans strains capable of hyphal formation (24), and acti-
vation 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 mole-
cule, 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

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-􏰂-1,2-oligomannoside 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. Ad-
herent 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 􏰇g/ml streptomycin, and 50 􏰇g/ml penicillin. Before
use, cells were gently scraped off with a rubber policeman and, depend-
ing on the experiment, either plated into eight-well Labtek tissue cul-
ture 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 phos-
phate-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-􏰂-1,2
oligomannoside monoclonal antibody AF1 as described previously (28).
PLM from C. albicans was prepared by extensive purification parti-
tion and hydrophobic interaction steps as described previously (26). The
structure of this molecule was determined by a combination of metha-
nolysis/HPLC, phosphorus/proton NMR, and ion spray mass spectrom-
etry 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 Stimula-
tion�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 􏰇l of boiling 2􏰄
concentrated electrophoresis sample buffer (1􏰄 electrophoresis sample
buffer: 125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 1% 􏰂-mercap-
toethanol, and bromphenol blue). Lysates were collected and clarified
by centrifugation for 10 min at 12,000 􏰄 g at 4 °C.

Full Article:

Keywords: Candida albicans Phospholipomannan Phagocytosed Yeasts Modulation Phosphorylation Macrophage Apoptosis drjefftop advanced

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