Laboratoire de Mycologie Fondamentale et Appliquée, INSERM EPI 9915, Université de Lille II, Faculté de Médecine H. Warembourg, Pôle Recherche, 59037 Lille Cedex, France
Correspondence: 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. E-mail: tjouault{at}univ-lille2.fr
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Key Words: MAP kinases • phagocytosis • yeast • pathogenicity • macrophages
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The host-parasite interactions that allow C. albicans to switch from a commensal to a pathogen that is capable of infecting a variety of tissues have been the subject of numerous studies and excellent recent reviews. Phagocytosis of C. albicans by mononuclear phagocytic cells is an important step in the control of infection, but phagocytosis and macrophage candidacidal activity are not always correlated [1 ]. It has been shown that although mononuclear phagocytic cells ingest C. albicans and the less pathogenic species Candida parapsilosis at similar rates, C. albicans strains are not killed as efficiently by monocytes because they have a better resistance to myeloperoxidase-derived oxidants [2 3 4 ]. Recent examination of internalization of C. albicans by macrophages has shown that the phenomenon is dependent on protein kinase C (PKC) and is independent of opsonization and macrophage mannose receptors. It involves the rapid recruitment of late endosomes and lysosomes, favoring the development of filamentous forms liable to escape the phagocytic cell [3 ].
Macrophage effector functions, especially those induced in response to
microbial stimuli, are known to be dependent on tyrosine
phosphorylation processes [5
]. This involves activation
of members of the mitogen-activated protein kinase (MAPK) family, which
consists of extracellularly regulated kinases (ERKs), stress-activated
protein kinases (SAPKs), and p38MAPK. All of these transduction
pathways are hierarchical cascades originating at the cell membrane
with receptors that recruit the small guanosine triphosphatase Ras. Ras
activates raf, a serine threonine kinase, which activates MAPK/ERK
kinases (MEKs). MEK, in turn, phosphorylates and activates ERK1 and
ERK2. Finally, ERK1/2 is able to phosphorylate both Elk1 and
90-kDa-ribosomal S6 kinases (p90RSK), which translocate to the nucleus.
Ras may also initiate parallel pathways leading to phosphorylation of
SAPK, p38MAPK, and I
B kinase.
Strategies to counteract host defense mechanisms by interfering with signal transduction pathways involved in endocytosis and phagocytosis have been developed by several intracellular pathogens [6 7 8 ]. Survival of the intracellular protozoan parasite Leishmania donovani is correlated with the activation of phosphotyrosine phosphatase, which in turn attenuates MAPK signaling [9 ]. The enteropathogenic bacterium Yersinia enterocolitica causes down-regulation of JNK (c-Jun N-terminal kinase), p38MAPK, and ERK1/2 in infected macrophages through the delivery of a set of outer proteins [8 ].
Among the different pathways involved in the infection of macrophages [10 ], activation of ERK1/2 was shown to be involved in the uptake of Listeria monocytogenes, which decreased in parallel with an overexpression of MAPK phosphatase (MKP)-1, an MEK-specific phosphatase [11 ]. The role of signal transduction in the internalization of yeasts by macrophages is poorly understood. In a recent study, incubation of yeasts with macrophages was demonstrated to result in signal transduction involving phosphotyrosine, which initiated proinflammatory cytokine production [12 ]. In this study, both pathogenic and nonpathogenic yeasts were used to investigate the pathways linked to yeast phagocytosis, the regulation of events leading to killing of yeasts, and the mechanisms involved in resistance of yeasts to killing by macrophages. In contrast to the nonpathogenic yeast Saccharomyces cerevisiae, which was phagocytosed and killed rapidly, internalized C. albicans survived in macrophages via a mechanism involving the signaling pathway, leading to p90RSK activation.
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Cell culture
The mouse macrophage-like cell line J774 (ECACC 85011428) was
derived from a tumor of a female BALB/c mouse. Adherent J774 cells were
cultured at 37°C in an atmosphere containing 5% CO2 in
Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented
with 10% heat-inactivated fetal calf serum (Valbiotech, Paris,
France), 5 mM L-glutamine, 100 µg/mL of streptomycin, and
50 µg of penicillin.
Yeasts
C. albicans VW32 (serotype A) and S.
cerevisiae SU1 [13
] were used throughout the study.
Both yeasts were maintained on Sabouraud dextrose agar (SDA) at 4°C.
Before the experiments, the yeast cells were transferred onto fresh SDA
and incubated at 37°C. After 20 h, the yeast cells were
recovered and washed with phosphate-buffered saline (PBS) (10 mM
PO4, pH 7.4).
Coculture of yeast cells with mammalian cells
The J774 cells were gently scraped with a rubber policeman and
distributed into 12-well culture dishes at a concentration of
106 cells per well. After 18 h, the adherent cells
were washed with culture medium and incubated with the yeasts. A ratio
of 20 yeast cells per J774 cell was chosen because it was shown to be
optimal for yeast interaction with these cells [14
].
After incubation for various periods, the cultures were washed with
DMEM to remove unbound yeast cells and then prepared for either
biochemical analysis or fungicidal assays. In some experiments, the
J774 cells were incubated for 1 h with 50 µM MEK inhibitor
PD98059 (New England Biolabs) before addition of the yeasts.
Fungicidal assays
After a 30-min incubation at 37°C in 5% CO2 with
yeast cells, the J774 cells were washed with DMEM to remove free yeast
cells and then recultured 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 for 10 min and were counted.
Recovered yeast cells were diluted in PBS, and a volume corresponding
to 100 individual yeast cells was plated onto SDA. After incubation for
24 h, the number of colony-forming units was determined.
Extraction and Western blotting
After coculture, the 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 of boiling twofold-concentrated
electrophoresis sample buffer [125 mM Tris-HCl (pH 6.8)], sodium
dodecyl sulfate (2%), glycerol (5%), ß-mercaptoethanol (1%), and
bromophenol blue). Lysates were collected and clarified by
centrifugation for 10 min at 12,000 g and 4°C.
Extracted proteins were separated by sodium dodecyl-sulfate-10% polyacrylamide gel electrophoresis before they were blotted onto a nitrocellulose membrane (Protran; Schleicher and Schuell, Dassel, Germany) for 2 h at 200 mA in a semidry transfer system. After being stained 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% bovine serum albumin (BSA) for 1 h at 20°C. Membranes were probed either with the phosphospecific antibodies (diluted 1:1,000) in TNT–1% BSA overnight at 4°C or with the anti-MKP antibody in TNT-1% milk for 1 h at 20°C. After being washed several times with TNT–1% BSA, the membranes were incubated for 1 h at 20°C with a 1:2,000 dilution of either HRP-conjugated anti-rabbit IgG in TNT-BSA or HRP-conjugated anti-mouse IgG in TNT-1% milk. After being washed, the membrane was incubated with enhanced chemiluminescence (ECL) detection reagents (SuperSignal Chemiluminescent Substrate; Pierce, Rockford, IL) and exposed to hyperfilm ECL.
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/).
Statistical analysis
All experiments were repeated at least four times. Values for
yeast viability are reported as means ± SD of results
from four different experiments. Statistical significance was
determined with Student’s t- test, and P <
0.05 was considered to be significant.
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 View larger version (29K): [in a new window] |
Figure 1. Phagocytosis of yeast cells by macrophages stimulates ERK but not
p38MAPK or the SAPK/JNK signal pathway. J774 cells were either
untreated or incubated with S. cerevisiae (S.c.) or C.
albicans (C.a.) blastoconidia. After 15 min (lanes 1) or 60 min
(lanes 2), the cells were lysed as described in Materials and Methods.
Whole-cell lysates were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. (A) Blots were probed with antibodies
specific for phosphorylated forms of SAPK/JNK, p38MAPK, or ERKs. (B)
Blots were incubated with antibodies specific for Elk1 or p90RSK, the
main downstream products of ERKs. Blots were developed with ECL, and
the autoradiograms were scanned. The data shown are representative of
four independent experiments. The results presented in the histogram
are the mean band density in arbitrary units as quantified by
densitometry analysis.
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Preferential induction of p90RSK activation by yeast phagocytosis
Because phosphorylation of both p90RSK and Elk1 by ERK1/2 is well
established [15
], the downstream pathway initiated after
phagocytosis of both yeasts was investigated. As shown in Figure 1B
,
ERK activation after ingestion of either C. albicans or
S. cerevisiae led to the specific activation of p90RSK in
the absence of phosphorylation of Elk1. A 2.8-fold increase in
phosphorylation of p90RSK compared with unstimulated cells was detected
after 15 min of ingestion of S. cerevisiae blastoconidia,
and phosphorylation decreased thereafter. With C.
albicans blastoconidia, a lower phosphorylation of p90RSK was
obtained 15 min after phagocytosis. This finding corresponded to a 52%
lower intensity than the p90RSK phosphorylation obtained when cells had
ingested S. cerevisiae blastoconidia. The phosphorylation
was nevertheless observed after 60 min of incubation, which was not the
case when macrophages had ingested S. cerevisiae
blastoconidia.
C. albicans can resist being killed by macrophages
Dysregulation of ERK1/2 phosphorylation has been proposed as a
mechanism used by different intracellular microorganisms to escape
uptake, depending on the phagocytic pathway. The effect of
dysregulation of ERK-dependent signaling on the ability of C.
albicans to resist phagocytosis was therefore investigated. Cells
were incubated with yeast blastoconidia and, after being washed, the
cells were lysed to recover ingested cells. Viability of endocytosed
yeasts was estimated by determining the number of colony-forming units
after 24 h of incubation. Figure 2
shows the results obtained with yeasts recovered 120 min after
incubation with the macrophages. A difference in yeast resistance was
apparent. More than 80% of recovered C. albicans
blastoconidia survived phagocytosis, whereas 80% of S.
cerevisiae blastoconidia were killed.
 View larger version (13K): [in a new window] |
Figure 2. Comparison of yeast survival after phagocytosis by J774 cells.
C. albicans or S. cerevisiae blastoconidia were
incubated with J774 cells at a concentration of 20 yeast cells
per J774 cell. After 30 min at 37°C, the free yeast cells were
removed by washing. Culture incubation was continued, and after 90 min
the endocytosed yeast cells were recovered by lysis of J774 cells with
sterile water. Recovered yeast cells were counted, and 100 cells were
transferred onto SDA. The number of colony-forming units (cfu) was
recorded after 24 h. Results presented are expressed as the
mean ± SD of four independent experiments.
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Treatment of macrophages with PD98059 inhibits killing of S.
cerevisiae blastoconidia
ERK phosphorylation depends on MEK, an upstream activator that is
phosphorylated by Raf [15
]. PD98059, a specific
inhibitor of MEK that inhibits signal transduction from MEK to ERK, was
used to examine whether inhibiting ERK signaling could lead to a
decrease in the susceptibility of yeasts to phagocytosis. Cells were
incubated with or without PD98059 before yeast blastoconidia were
added. We first examined the effect of the treatment on the signal
induced by ingestion of yeasts. As shown in Figure 3
, a 37% decrease in ERK phosphorylation was observed in response
to both C. albicans and S. cerevisiae after
treatment of cells with 50 µM PD98059. The effect of PD98059 on the
downstream product p90RSK with both yeasts was also significant.
However, a greater inhibitory effect on the signal was observed after
C. albicans ingestion (75% inhibition of signal induced by
C. albicans compared with 51% inhibition obtained with
S. cerevisiae).
 View larger version (51K): [in a new window] |
Figure 3. Effect of PD98059 inhibitor on phosphorylation of MEK, ERK, and p90RSK.
J774 cells were pretreated without or with 50 µM PD98059 inhibitor
for 1 h at 37°C before addition of C. albicans or
S. cerevisiae blastoconidia. After 15 min of coculture at
37°C, the J774 cells were extracted, and the proteins were examined
by Western blotting with anti-MEK, anti-ERK, or anti-p90RSK
phosphospecific antibodies. The autoradiograms presented are
representative of four independent experiments.
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 View larger version (17K): [in a new window] |
Figure 4. Effect of PD98059 inhibitor on yeast survival after phagocytosis. J774
cells were untreated (open bars) or pretreated (closed bars) with 50
µM PD98059 inhibitor for 60 min at 37°C. C. albicans or
S. cerevisiae blastoconidia were then added [at a 1:20
(cell/yeast cell) ratio] and cultured with the cells for 30 min. Free
yeasts were discarded, and after 90 min the endocytosed yeast cells
were recovered by lysis of J774 cells, and 100 yeast cells were
transferred onto SDA. The number of colony-forming units (cfu) was
scored after 24 h. Results presented are means ±
SD of four independent experiments.
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Activation of phosphatase MKP-1 after ingestion of C.
albicans by macrophages
The hypothesis that dysregulation of ERK-p90RSK phosphorylation
was due to induction of MKP-1 activation by C. albicans in
macrophages was investigated. MKP-1 has been shown to be one of the
main phosphatases involved in the regulation of the ERK signaling
pathway. Figure 5
shows the results obtained when extracts from macrophages were
immunostained with an mAb specific for the activated form of MKPs.
Compared with control cells, high-level activation of MKP-1 was
detected 15 min after ingestion of both yeasts (4.6-fold and 3.9-fold
increases for C. albicans and S.
cerevisiae, respectively). However, although
amplification of the MKP-1 signal with S. cerevisiae
decreased and returned to an intensity equivalent to that obtained with
control cells, the activation was maintained and even increased
(9.7-fold increase compared with control cells) after 60 min of
ingestion of C. albicans blastoconidia.
 View larger version (39K): [in a new window] |
Figure 5. Induction of MKP-1 after phagocytosis of C. albicans
blastoconidia. J774 cells were incubated without (None) or with either
C. albicans (C.a.) or S. cerevisiae (S.c.)
blastoconidia for 15 min (lanes 1) or 60 min (lanes 2) at 37°C. The
cells were extracted, and extracted proteins were revealed by Western
blotting with anti-MKP antibodies. Autoradiograms were scanned, and the
band density was measured. The results presented are from one
experiment representative of three independent experiments.
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Several reports have focussed on the role of the different signal transduction pathways in activation of phagocytic cells. Most of these studies have been concerned with the role of MAPK in neutrophil activation. For example, it has been shown that, if ß-glucan- and IgG-opsonized bacteria induce high-level phosphorylation of both ERK1/2 and p38MAPK in the absence of SAPK/JNK activation, unopsonized bacteria stimulate a minimal increase in ERK1/2 and p38MAPK activity [7 ]. p38MAPK, together with phosphatidylinositol 3-kinase, also participates in signaling pathways leading to NADPH oxidase activation, the main source of O2-, and in signaling induced during ß-glucan-dependent phagocytosis [19 ]. Although MEK, as the upstream activator, has great importance in the response of neutrophils [20 ], Hii et al. [6 ] showed by using the MEK inhibitor PD98059 that the ERK cascade plays only a minor role in the microbicidal activity of these cells and that PD98059 has no effect on cell adherence or degranulation. For macrophages, the involvement of individual MAPKs in the different functions of the cell is not yet clear. The roles of both p38MAPK and SAPK/JNK in cytokine production are well established [21 , 22 ]. Activation of MEK/ERK has been shown to be critical for cytokine production and also for prostaglandin E2 production in response to lipopolysaccharide [10 ]. Besides its involvement in the control of cell proliferation [23 ], ERK activation has been shown to play a central part in the response to hypotonic stress [24 ] or to reactive oxygen species [25 , 26 ]. This is important because reactive oxygen species have an important relevance in macrophage microbicidal activities [20 ] and spreading [27 ]. The role of the MEK/ERK pathway in the phagocytic function of macrophages has been mainly revealed by evidence of the interference between pathogens and the signal transduction pathways involved in phagocytosis to counteract the host defense mechanisms. Large numbers of microorganisms have targeted the MEK/ERK signal transduction pathway to deactivate macrophage functions and escape phagocytosis. This pathway has been found to be of major importance in response to different microbial stimuli like bacteria [7 , 8 , 11 , 28 ] or bacterial lipopolysaccharide [10 , 29 , 30 ]. We have shown that endocytosis of yeasts by macrophages is accompanied by a signal resembling the one already described for other microorganisms involving activation of the MEK/ERK pathway. However, the results presented here demonstrate a major difference for yeast endocytosis, which consists of the preferential phosphorylation of p90RSK instead of the activation of Elk1. The involvement of p90RSK in the macrophage response to yeasts leading to killing was also suggested by the effect of PD98059 treatment, which led concomitantly to decreased ERK/p90RSK phosphorylation and to survival of the sensitive yeast S. cerevisiae.
Recently, p90RSK has been shown to phosphorylate the
Na+/H+ exchanger isoform-1, which is a key
member of a family of exchangers that regulates intracellular pH and
cell volume [31
]. p90RSK is also involved in
stimulus-induced targeting of IB
phosphorylation at serine 32 and
serine 36 in response to cell stimulation by different stimuli, leading
to its rapid degradation and to the translocation of large pools of
NF-B to the nucleus [32
]. p90RSK stimulates binding
of Bad to 14-3-3, thus blocking Bad-mediated cell death in a
serine-112-dependent manner; serine-112 of the protein Bad is a site
known to regulate apoptotic cell death by interleukin-3
[33
].
Although C. albicans is not considered to be a true intracellular pathogen, it harbors the ability to impair macrophage lytic activities, which resembles the capability already described for bacteria and other parasites. Recent studies have shown that C. albicans was able to escape from endocytic vesicles by interfering at the time of their acidification [3 ]. Here, we report that ingestion of C. albicans by macrophages was accompanied by a decrease in phosphorylation of pathways that are engaged in the cell response to microbial agents. Different mechanisms have been described for microbes that escape from macrophage lytic activities. Among these, overexpression of phosphatases such as MKP-1, one of the MAPK phosphatases described for its regulatory activity on the MEK-ERK pathway [34 ], has been involved in the dysregulation of macrophage activities, thus allowing the survival of L. monocytogenes [11 ]. A role for this phosphatase in C. albicans-induced dysregulation of the ERK pathway was suggested because after phagocytosis of C. albicans, an intense activation of MKP-1 occurred.
The exact mechanism of activation of MKP-1 by C. albicans remains to be identified, and the characterization of the molecules involved in this process is underway. However, the parallel induction of phosphatase expression and the decreased phosphorylation of both ERK and p90RSK after endocytosis of C. albicans, together with the survival of this yeast, suggest a possible involvement of these processes in yeast escape.
Received October 19, 2000; revised February 5, 2001; accepted February 6, 2001.
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