EUKARYOTIC CELL, Apr. 2007, p. 658–663
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 6, No. 4
Drosophila melanogaster Thor and Response to Candida albicans Infection?†
A. Levitin,1* A. Marcil,1G. Tettweiler,2M. J. Laforest,1U. Oberholzer,1
A. M. Alarco,1D. Y. Thomas,3P. Lasko,2and M. Whiteway1,2
Genetics Group, Biotechnology Research Institute, National Research Council, Montreal, Quebec H4P 2R2, Canada1;
Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada2; and
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada3
Received 31 October 2006/Accepted 23 January 2007
We used Drosophila melanogaster macrophage-like Schneider 2 (S2) cells as a model to study cell-mediated innate
immunity against infection by the opportunistic fungal pathogen Candida albicans. Transcriptional profiling of S2
during this interaction is the D. melanogaster translational regulator 4E-BP encoded by the Thor gene. Analysis of
Drosophila 4E-BPnullmutant survival upon infection with C. albicans showed that 4E-BP plays an important role in host
defense, suggesting a role for translational control in the D. melanogaster response to C. albicans infection.
Candida albicans is a part of normal microbial flora that can be
found in mucocutaneous surfaces of the oral cavities, gastrointes-
tinal tracts, and vaginas of many mammals, including humans.
Although C. albicans does not normally cause severe disease in
immunocompetent hosts, this pathogen can trigger life-threaten-
ing systemic infections in immunocompromised individuals.
Mammals respond to C. albicans infection through activating
both acquired and innate immune responses (3, 30), with the
innate immune response as the first defense. Since the innate
immune response is evolutionarily highly conserved, Drosophila
melanogaster is a promising system for studying virulence charac-
teristics of medically important pathogens such as C. albicans (7,
32). The Drosophila immune response is composed of both hu-
moral and cellular components (2). The innate immune system
consists of two major networks defined by the Imd (immune
deficiency) and Toll pathways that are activated by fungal and
bacterial infections (7). These pathways initiate humoral antimi-
crobial defenses in the Drosophila fat body, the analog to a mam-
malian liver. The cellular immune response involves plasmato-
parasites; transcription activation of a variety of pathways is nec-
essary for these responses.
In eukaryotes, regulation of gene expression at the transla-
tional level is a very complex process. It allows for very rapid
adaptive changes in global protein synthesis levels and for
selective mRNA translation during the regulation of the cell
cycle, development, apoptosis, the response to cell prolifera-
tion conditions, and cellular stress conditions, such as infec-
tion. During translation initiation, the 40S preinitiation com-
plex is recruited to mRNA by interactions with the cap-binding
complex eIF4F (eukaryotic initiation factor 4F). The eIF4F
complex consists of 3 subunits: eIF4E, the cap binding protein;
eIF4A, a RNA helicase; and eIF4G, a scaffolding protein.
The activity of eIF4E is regulated by the eIF4E-binding pro-
teins (4E-BPs). These repressor proteins inhibit cap-dependent
translation by preventing the association of eIF4E with eIF4G
and thereby suppressing the formation of the cap-binding com-
plex. The binding of 4E-BPs to eIF4E is modulated by the phos-
phorylation status of the 4E-BPs at several serine and threonine
residues. Under active growth conditions, 4E-BPs are hyperphos-
phorylated, remain dissociated from eIF4E, and are inactive in
blocking cap-dependent translation. However, under conditions
that block cell proliferation or induce apoptosis, hypophosphor-
ylated 4E-BPs sequester eIF4E and inhibit cap-dependent, but
not cap-independent, translation (9, 13, 15, 34).
Recent studies have shown that Drosophila has a single
d4E-BP (21), in contrast to mammals, which express three
distinct 4E-BP proteins (24, 26). Drosophila 4E-BP is an effec-
tor of cell growth (21). The phosphorylation of d4E-BP is
stimulated by insulin via the conserved insulin receptor (dInR-
PI3K-Akt-TSC-dTOR) pathway. In starved Drosophila S2
cells, most of the d4E-BP consists of the nonphosphorylated
isoform (?), which is active in binding deIF4E. Treatment with
insulin induces a shift to another isoform (?), hyperphosphor-
ylated at Thr37 and Thr46, which causes d4E-BP dissociation
from deIF4E (20).
Among the first-line defense players of the innate immune
system are the macrophages, which can phagocytose patho-
gens. In the present work, we used S2 cells, which share many
characteristics with mammalian macrophage cells, to study
pathogen-host interactions. It was recently described by Stros-
chein-Stevenson et al. (32) that S2 cells engulf C. albicans as
early as 30 min after they encounter each other. We showed
that phagocytosis of C. albicans cells induces differential ex-
pression of immune response genes. Microarray analysis of the
host-pathogen interaction identified several genes involved in
innate response to C. albicans infection, including Thor, which
encodes d4E-BP. Subsequently, we investigated the impor-
tance of d4E-BP in vivo and observed an increased sensitivity
of d4E-BPnullflies to Candida infection. Our data suggest that
d4E-BP is important for fly survival after Candida infection.
MATERIALS AND METHODS
Yeast, bacterial, and Drosophila strains and cell lines. The C. albicans strains
used in this study were SC5314 (12) and CAI4-GFP, expressing a soluble intra-
* Corresponding author. Mailing address: 6100 Royalmount Ave,
Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6146. Fax:
(514) 496-6213. E-mail: email@example.com.
† This is National Research Council publication 47513.
?Published ahead of print on 2 February 2007.
cellular green fluorescent protein (GFP) (ura3::imm434/ura3::imm434 pAM5.6)
(2, 10). These strains were grown in YPD (1% yeast extract, 2% peptone, 2%
dextrose, 0.05% uridine, pH 5.5) or SD-ura (0.15% dropout uracil, 0.05% uri-
dine, 0.67% yeast nitrogen, 2% dextrose) media, respectively. Drosophila Schnei-
der 2 (S2) cells (Invitrogen) were grown in Schneider’s media (Invitrogen)
supplemented with 10% heat-inactivated fetal bovine serum (S-10 medium)
according to the supplier’s specifications (ATCC). The d4E-BPnull(Thor2), re-
vertant (Thor1Rv1), and Oregon-R wild-type flies are as described previously (5,
6, 33). The Saccharomyces cerevisiae strain used was MLY40 (17).
Time-lapse microscopy. The day before introducing the fungal cells, 106Dro-
sophila S2 cells were seeded in a Bioptechs petri dish. S2 cells were then incu-
bated with live or 4% paraformaldehyde-fixed Candida cells or with 3.53-?m
latex beads (Estapor Microspheres). Phase-contrast as well as epifluorescence
pictures were taken at a ?400 magnification every 15 min with a DMIRE2
inverted microscope (Leica Microsystems Canada) equipped with a Hamamatsu
cooled charge-coupled-device camera, a Bioptechs temperature-controlled stage
adapter, and a Ludl motorized stage. Openlab software (Improvision) was used
for image acquisition.
Immunofluorescence. Drosophila S2 cells (107) were seeded in six-well plates
(Becton-Dickinson), and Candida cells (strain CAI4-GFP) were added at a
multiplicity of infection (MOI) of 1. At the indicated incubation time, cells were
washed two times in S-10 medium and stained with an anti-Candida antibody as
previously described (29), except the secondary antibody was a Rhodamine
red-X-conjugated F(ab)?2 donkey anti-rabbit antibody (Jackson Immuno-
Research Laboratories Inc., West Grove, PA) diluted 1:200 in S-10. Epifluores-
cence was monitored using the appropriate filters, at ?400 magnification.
Total RNA and mRNA extractions. Total RNA was extracted by the hot
phenol extraction method (8). mRNA isolation was performed using the Micro-
FastTrack mRNA isolation kit from Invitrogen according to the manufacturer’s
Microarrays. The microarrays used in this study were purchased from the
Drosophila Microarray Center (d12k v1) (23). Transcription profiles for each
condition represent the average of at least 4 to 9 independent hybridizations.
These include dye swap hybridizations (Cy3/Cy5 and Cy5/Cy3) from at least
three independently produced RNA preparations. The DNA microarray slides
were scanned with a ScanArray 5000 scanner (version 2.11; GSI Lumonics, then
Packard BioScience, now Perkin Elmer-Cetus, Wellesley, CA) at a 10-?m res-
olution. Quantitation and normalization of DNA microarrays were performed as
described previously (22). The resulting 16-bit TIFF files were quantified with
QuantArray software (versions 2.0 and 3.0; Perkin Elmer-Cetus). Statistical
analysis and visualization were performed with GeneSpring software (Silicon
Genetics, Redwood City, CA) as described previously (22).
Northern blot analyses. The Northern blot analyses were performed as de-
scribed previously (18). Probes for d4E-BP and RpL4 genes were synthesized as
follows. Fragments from the d4E-BP (Thor) gene were amplified by PCR using
a forward primer, 5?-TGGGGACGGGCACGCACTTG-3?, and a reverse
primer, 5?-GTGGTCCCCTGGTGGTCT-3?. The RpL4 probe was used as an
internal control to monitor RNA loading and transfer. Fragments for the RpL4
gene were amplified using a forward primer, 5?-GGCGGCGACCTTCTTCTT-
3?, and a reverse primer, 5?-GTGTGCCGACAGCTAGGATT-3?.
Antibodies and Western blot analyses. Anti-d4E-BP was a generous gift from
N. Sonenberg (21). Anti-phospho-4E-BP1 (Thr37/46) antibodies were obtained
from Cell Signaling Technology, Inc. Anti-actin antibodies were obtained from
Protein extracts (50 ?g) were loaded on a 15% acrylamide gel, separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose membranes (Bio-Rad) for Western blot analysis. Membranes were
incubated with anti-d4E-BP primary antibody (1:2,000) in Tris-buffered saline
containing 0.05% Tween 20 plus 5% bovine serum albumin or with a 1:2,000
dilution of anti-actin monoclonal antibody, followed by a 1:2,000 dilution of
anti-rabbit or anti-mouse horseradish peroxidase-conjugated immunoglobulin G
(Santa Cruz Biotechnology). The proteins were detected using Lumi-Light West-
ern blotting substrate (Roche).
Drosophila infection. Infection of flies (1- to 3-day-old virgin females or males;
30 per experimental group) was performed as described previously (16) with a
thin needle dipped in a concentrated cell pellet containing 200 optical density of
the yeast cells used in our study. The inoculum size was evaluated at approxi-
mately 103cells per fly. Following infection, flies were maintained at 25°C on
regular fly medium. Infection experiments were performed at least three inde-
pendent times, and standard deviations were calculated.
Phagocytosis of Candida albicans by S2 cells. Drosophila
plasmatocytes are responsible for the phagocytosis and de-
struction of apoptotic cells and microorganisms (11, 19). The
plasmatocytes share several properties with mammalian mac-
rophages at the structural and molecular levels (1, 25). In this
study, we used S2 cells to analyze cell-mediated innate immu-
nity and phagocytosis. This cell line, which is derived from
Drosophila embryos, expresses macrophage-like genes (croque-
mort, dSR-CI, and PGRP-LC) and possesses macrophage-like
phagocytic properties (25, 28, 32). The goal of the present work
was to analyze the effect of the internalized Candida on S2
cells. To establish whether S2 cells phagocytize C. albicans, we
monitored the internalization of GFP-labeled C. albicans CAI4
cells (CAI4-GFP) in coincubation experiments with S2 cells for
up to 6 h (Fig. 1). The results indicated that C. albicans cells
were indeed engulfed by S2 cells. We also took a double-
labeling approach to distinguish between C. albicans cells in-
ternalized versus attached to the surface. Figure 2 shows that
Candida cells associated with S2 cells are efficiently internal-
ized, with the percentage of engulfed Candida increasing with
the incubation time. It is noteworthy that S2 cells are loosely
adherent and many cells are lost during the immunofluores-
Endpoint dilution analysis of Candida interaction with Dro-
sophila S2 cells. To assess the antifungal activity of Drosophila S2
cells, we conducted an endpoint survival assay where we moni-
tored the survival of Candida SC5314 and GFP-labeled CAI4
strains in the presence of Drosophila S2 cells. Survival was mea-
by the number of colonies in the absence of the S2 cells. In the
FIG. 1. Interaction of Drosophila S2 cells with Candida strain
CAI4-GFP. Drosophila S2 cells were incubated at 25°C with Candida at
an MOI of 1 and monitored by time-lapse microscopy at ?400 mag-
nification for the indicated times (bottom left, in hours). Arrows point
to representative Candida cells engulfed by the S2 cells.
VOL. 6, 2007DROSOPHILA S2 CELLS TO STUDY HOST-PATHOGEN INTERACTION659
presence of Drosophila S2 cells, the number of colonies formed
was 57.4% ? 9.2% lower in the case of strain SC5314 and 61.3%
? 4.5% lower in the case of strain CAI4-GFP than the number of
Candida cells in the absence of S2 cells.
Transcriptional analysis of S2 cells’ response to the pres-
ence of C. albicans. We used high-density microarrays repre-
senting 10,500 Drosophila genes to study global gene expres-
sion changes of the S2 cells in response to C. albicans infection.
Drosophila S2 cells were coincubated with the C. albicans wild-
type strain SC5314, at a starting MOI of 1, in Schneider me-
dium supplemented with 10% fetal bovine serum at 25°C. A
comparison of mRNA levels at 3 h and 6 h after infection,
respectively, revealed relatively few changes in gene expres-
sion. In Table 1, we show a list of 27 genes differentially
regulated by the presence of C. albicans in S2 cells (with a
cut-off of 1.5-fold at 6 h postinfection). Among the transla-
tional repressors, Thor was one of the most strongly induced
genes in the presence of C. albicans (5.6-fold after a 6-h treat-
ment). A representative of secreted proteins, lox (lysyl-oxydase
like or dLOXL-1) was observed to be induced 4.5-fold at the
same time point. A member of the immunoglobulin superfam-
ily, the Impl2 (CG15009) gene was also induced 2.6-fold as well
as the fok gene (3.3-fold). A negative regulator of translation,
poly(A)-binding protein-interacting protein 2a (4, 31), was also
induced 1.5-fold after a 6-h Candida infection. Attacin A, en-
coding an antibacterial peptide, showed a 1.6-fold up-regula-
tion. Among detoxification or stress-related proteins in S2 cells
infected with C. albicans, the Hph gene was up-regulated 1.6-
fold. We also observed the up-regulation of genes involved in
sterol and lipid metabolism, such as Fpps (2.2-fold) and ifc
(1.6-fold). Four genes were down-regulated upon Candida in-
fection of S2 cells. The String gene involved in the mitotic cell
cycle was down-regulated 1.8-fold in 6 h upon infection. A
myoblast fusion gene, rolling stone, was also down-regulated
1.7-fold, along with an exonuclease-like gene and an unknown
gene, CG30457 (down-regulated 1.6- and 1.5-fold, respec-
d4E-BP mRNA and protein levels increase upon C. albicans
infection. Northern blot analyses were performed to confirm
the induction of d4E-BP detected by the microarray analyses.
As shown in Fig. 3, d4E-BP mRNA levels increase in the
presence of live C. albicans; up-regulation of d4E-BP expres-
sion is not detected with coincubation with latex beads or with
fixed C. albicans cells. Therefore, the induction of d4E-BP
expression appears specific to the immune function in S2 cells
and not to the phagocytosis of fixed pathogen or latex particles.
We also analyzed the presence of the d4E-BP protein in
total extracts from S2 cells and C. albicans-coincubated cells
(Fig. 4). Consistent with our microarray and Northern blot
results, we observed increased levels of d4E-BP protein after a
6-h coincubation with live C. albicans. Using a phosphospecific
antibody, we found that most of the increased d4E-BP was in
the hypophosphorylated (?), active form, although a small
increase in the hyperphosphorylated (?) form of d4E-BP was
also observed. These results establish that, in S2 cells in the
presence of live C. albicans, active d4E-BP protein is present at
a higher level.
Response of d4E-BPnullflies to infection by C. albicans. To
determine whether d4E-BP contributes to antifungal immu-
nity, we analyzed the survival of Drosophila 4E-BPnullmutant
flies following their infection with C. albicans. In separate
experiments, 1- to 3-day-old male and female virgin flies were
pricked with a very thin needle coated with concentrated C.
albicans SC5314 or S. cerevisiae strain pellets or with a sterile
needle as a negative control. We measured the survival of
Oregon-R, revertant (Thor1rev1), and d4E-BPnull(Thor2) flies
6 h after infection (Fig. 5). We found that d4E-BPnullmutant
flies are about 50% less resistant to Candida infection than
controls, indicating that d4E-BP is involved in conferring im-
munity to this fungal species. Although d4E-BPnullmutant flies
showed a decreased resistance to Candida infection in both
FIG. 2. Phagocytosis of Candida strain CAI4-GFP by Drosophila S2
cells. Drosophila S2 cells were incubated at 25°C with Candida CAI4-
GFP at an MOI of 1 for the indicated time. They were then stained
with an anti-Candida polyclonal antibody (in red), as described in
Materials and Methods. Engulfed Candida, protected from primary
antibody binding, remained green, whereas nonphagocytosed Candida
660 LEVITIN ET AL.EUKARYOT. CELL
sexes, it is noteworthy that female flies showed a higher resis-
tance than the males. In contrast, the d4E-BPnullmutation had
no effect on survival after infection with S. cerevisiae.
We used hemocyte-like Drosophila S2 cells, in coculture
experiments with C. albicans, as a model representing cell-
mediated innate immunity against infection by this pathogen.
The transcriptional profiling of the S2 cells during the phago-
cytosis of C. albicans revealed a number of differentially ex-
pressed Drosophila genes in response to the pathogen. Eight of
TABLE 1. Genes regulated in S2 cells in the presence of Candida albicans strain SC5314a
CG no.FlyBase Full nameGene product
ratio at time (h):
P value (6 h)
eIF4E binding protein
Lysyl oxidase and Scavenger receptor
cysteine-rich (SRCR) domains
Cell adhesion, extracellular
Cholesterol metabolism (EC 188.8.131.52)
cascade/autophagy cell death
domain/diphtheria toxin resistance
HMG-1, HMGY DNA binding
Sphingolipid delta-4 desaturase
Similar to yeast Cwfj/RNA splicing
L-Amino acid transporter
Amino acid/polyamine transporter
Negative regulator of translation
Fledgling of Kpl38B
Ecdysone-inducible gene L2
FppsFarnesyl pyrophosphate synthase
HphHIF prolyl hydroxylase 1.2
CG17836 1.3 1.60.0286
ifcInfertile crescent 1.3
CG3767 JhI-26Putative CHK domain (choline
Alpha/beta hydrolase flod/aromatic
Glutathione transferase (EC 184.108.40.206)
Glutathione transferase (EC 220.127.116.11)
GTP binding domain/putative
phosphatase (EC 3.1.3)
3?–5? exonuclease and RNase H-like
Glutathione S-transferase E9
Glutathione S-transferase E8
CG1395 stg String
aGenes whose expression was either up-regulated (positive values) or down-regulated (negative values) in Candida-treated cells compared to the Candida-free S2
cells at indicated time points.
FIG. 3. Northern blot analysis of Thor gene induction in S2 cells.
Lanes from left to right: S2 cells (control), S2 cells infected with live
wild-type C. albicans (SC5314), S2 cells in the presence of paraformalde-
hyde-fixed C. albicans, and S2 cells ingesting latex beads, for 3 and 6 h.
FIG. 4. Western blot of d4E-BP protein in S2 cells. Lanes from left to
right: S2 cells alone (control), S2 cells infected with live C. albicans
(SC5314), S2 cells in the presence of paraformaldehyde-fixed C. albicans,
and S2 cells ingesting latex beads for 6 h. Identical amounts of total
protein (30 ?g) were analyzed by Western blotting with 1868 antibody to
d4E-BP or phospho-4E-BP1 (thr37/46). ?, active, nonphosphorylated iso-
form; ?, hyperphosphorylated isoform; actin, loading control.
VOL. 6, 2007 DROSOPHILA S2 CELLS TO STUDY HOST-PATHOGEN INTERACTION 661
these genes were up-regulated more than 2-fold after 6 h of
infection, and the remaining 19 genes were up- or down-reg-
ulated at least 1.5-fold. Among the differentially expressed
genes, there were known immune-related genes, such as AttA
or Thor, and several new candidates for genes involved in
cell-based immunity. However, the relatively small number of
genes modulated by the interaction with C. albicans indicates
that Drosophila S2 cells do not rapidly regulate the transcrip-
tion of a large number of genes in response to the presence of
this fungus. In particular, several known immune-related
genes, such as those encoding the antifungal peptides Droso-
mycin and Metchnikowin or other components of the Toll path-
way, are not represented in this profile. Either their regulation
might have occurred at times outside the scope of our exper-
iments, or S2 cells lack the capacity to transcriptionally regu-
late them. Interestingly, the strong and rapid induction of the
d4E-BP (Thor) gene suggests that regulation of translation
could be a significant mechanism in Drosophila cell-based im-
munity. Drosophila 4E-BP is homologous to 4E-BPs from
other species, and the phosphorylation sites in mammalian
4E-BP1 are conserved in d4E-BP (21). Recent studies have
shown that Drosophila has a single d4E-BP (21), in contrast to
mammals, which express three distinct 4E-BP proteins (24, 26).
This makes Drosophila an excellent model to study the function
of 4E-BP in the immune response to pathogens.
In addition to the Northern blots confirming the activation
of d4E-BP in S2 cells “infected” by live C. albicans (Fig. 3),
Western blot analysis (Fig. 4) confirmed the increase in
d4E-BP protein level. We have shown that this protein also
remains mostly in its active ? form in the S2 cells in the
presence of live C. albicans. Such an increase in the level of the
active hypophosphorylated d4E-BP protein would compete
with the formation of the cap-binding complex and inhibit
To date, the only transcription factor known to regulate the
transcription of d4E-BP is dFOXO, which is negatively regu-
lated by insulin and positively regulated by different cellular
stresses, through the forkhead response element in the d4E-BP
gene promoter (14, 33). However, an additional signaling path-
way might target d4E-BP in response to infection in flies, since
our transcriptional analysis also failed to detect a change in
expression of the FOXO gene (the gene was up-regulated by a
maximum of 1.1-fold at 6 h post-Candida infection) as well as
dInr, the insulin receptor gene positively regulated by FOXO
(27) (Table 1).
In this work, we have established the S2 cell line as a model
for the study of gene expression during host-Candida interac-
tion. We used transcriptional profiling to identify several can-
didates for new immune-related genes in Drosophila and
placed d4E-BP as an important player in defense against C.
albicans infection. In further studies, the signaling pathway
directing the expression and phosphoregulation of d4E-BP
in the Drosophila immune response should be of particular
We thank N. Sonenberg for the anti-d4E-BP antibodies.
This work was supported by Canadian Institutes of Health Research
(CIHR) grant to M.W. and D.Y.T. A.L. gratefully acknowledges a
Canadian Government Laboratory Visiting Fellowship.
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