Caspar, a suppressor of antibacterial immunity
Myungjin Kim*, Jun Hee Lee*, Soo Young Lee*, Eunhee Kim†, and Jongkyeong Chung*‡
*National Creative Research Initiatives Center for Cell Growth Regulation and Department of Biological Sciences, Korea Advanced Institute of Science
and Technology, 373-1 Kusong-dong, Yusong, Taejon 305-701, Korea; and†School of Bioscience and Biotechnology, Chungnam National University,
220 Gung-dong, Yusong, Taejon 305-764, Korea
Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved September 5, 2006 (received for review April 21, 2006)
Drosophila has a primitive yet highly effective innate immune
system. Although the infection-dependent activation mechanisms
of the Drosophila immune system are well understood, its inhib-
itory regulation remains elusive. To find novel suppressors of the
immune system, we performed a genetic screening for Drosophila
mutants with hyperactivated immune responses and isolated a
loss-of-function mutant of caspar whose product is homologous to
Fas-associating factor 1 in mammals. Interestingly, caspar mutant
flies showed increased antibacterial immune responses including
increased resistance to bacterial infection and a constitutive ex-
pression of diptericin, a representative antibacterial peptide gene.
Conversely, ectopic expression of caspar strongly suppressed the
infection-dependent gene expression of diptericin, which allowed
bacterial outgrowth. Consistent with these physiological pheno-
types, Caspar negatively regulated the immune deficiency (Imd)-
mediated immune responses by blocking nuclear translocation of
Relish, an NF-?B transcription factor. In addition, we further dem-
onstrated that Dredd-dependent cleavage of Relish, a prerequisite
event for the nuclear entry of Relish, is the target of the Caspar-
mediated suppression of the Imd pathway. Remarkably, Caspar
was highly specific for the Imd pathway and did not affect the Toll
pathway, which is crucial for antifungal immunity. Collectively, our
elucidation of an inhibitory mechanism of the Imd pathway by
Caspar will provide a valuable insight into understanding complex
regulatory mechanisms of the innate immune systems in both
Drosophila and mammals.
immune deficiency ? Relish
Drosophila is able to fight invading microorganisms via the innate
that exposes microbes to reactive oxygen; phagocytosis or encap-
sulation of invaders; and massive synthesis of cationic antimicrobial
peptides. The synthesis of antimicrobial peptides is induced in the
fat body (a functional analog of the mammalian liver) within a few
hours after injury or microbial infection, and the synthesized
peptides are immediately secreted into the insect hemolymph in
high concentrations. Molecular genetics studies have shown that
two distinct pathways, the immune deficiency (Imd) pathway and
antimicrobial peptides (1, 2, 4–6). The Imd pathway controls acute
expression of most antibacterial peptide genes like diptericin
through the mammalian p105?p110 NF-?B homolog Relish (7),
whereas the Toll pathway regulates gene expression of antifungal
peptides such as drosomycin through the mammalian p65 NF-?B
homologs Dorsal and Dorsal-related immunity factor (DIF) (8).
The Imd pathway was initially characterized by a mutation in the
imd gene, which blocks infection-dependent induction of antibac-
terial peptide genes and renders flies highly susceptible to Gram-
negative bacterial infection (4, 6). Extensive genetic screening of
Drosophila mutants with phenotypes similar to imd led to the
identification of a number of other genetic components related to
imd, and additional genetic and biochemical studies suggested a
attractive model for the study of innate immunity (1, 2).
mechanistic model of signal flow in the Imd pathway that is similar
to the mammalian TNF receptor signaling pathway (1, 2). The
pattern recognition receptors (9) stimulated by peptidoglycans
12), through dTAK1 (13) and dFADD (14, 15). Subsequently, the
DmIKK complex phosphorylates Relish (11, 16), and Dredd
caspase cleaves Relish into the N-terminal Rel homology domain
and the C-terminal inhibitory domain (17–19). This cleavage leads
to nuclear translocation of the Rel homology domain, which is
required for the induction of antibacterial gene transcription
with microbial infection, negative regulation of the immune signal-
ing pathway is crucial for preventing deleterious side effects of
unnecessarily induced or hyperactivated immune responses in an
organism. Previously, Khush et al. (20) showed that Skp1?Cullin?
F-box components in the ubiquitin–proteasome pathway have a
repressive role for antibacterial immune responses in Drosophila,
demonstrating the existence of a negatively regulating mechanism
in the immune signaling pathway.
To uncover other inhibitory mechanisms of antibacterial immu-
nity, we screened for Drosophila mutants with hyperactivated
immune responses using GenExel (Taejon, Korea) EP lines and
subsequently identified a mutant, caspar, which displayed a consti-
tutive expression of antibacterial peptides and showed resistance to
which has been previously reported to associate with various
components of the TNF?NF-?B signaling pathway, such as FAS,
roles of FAF1 have remained elusive because of the absence of a
genetic model. Here, using various biochemical and genetic tech-
niques, we clearly demonstrated that Caspar specifically suppresses
the Imd-mediated immune response by preventing Dredd-
dependent nuclear translocation of Relish in Drosophila.
Isolation of caspar Mutants. To discover suppressors of the Dro-
sophila immune system, we screened mutants with ectopic mela-
nization, which is generally observed in various mutants suffering
(24, 25). Among 15,000 independent nonlethal EP lines from the
GenExel library, we isolated a mutant line showing a high rate of
Author contributions: M.K. and J.C. designed research; M.K., J.H.L., and S.Y.L. performed
research; M.K. and E.K. contributed new reagents?analytic tools; M.K. analyzed data; and
M.K. and J.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: Imd, immune deficiency; DIF, Dorsal-related immunity factor; FAF1, Fas-
associating factor 1; hFAF1, human FAF1; UAS, ubiquitin-associated.
‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
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and named the putatively affected gene as ‘‘caspar’’ (named after
one of the three Magi, ‘‘Caspar’’) and the mutant allele as
‘‘casparP1.’’ The melanization of casparP1flies was conspicuous
around internal organs such as the gut and fat body (Fig. 1A).
casparP1Is Resistant to Bacterial Infection. To test whether casparP1
mutation enhances Drosophila immune responses against bacterial
infection, we infected wild-type and casparP1flies by pricking them
with a needle dipped in a concentrated solution of Gram-negative
wild-type flies showed a moderate decrease in viability upon
infection, key1flies, which carry a loss-of-function mutation in the
gene encoding the Drosophila IKK? homolog (kenny) (12), showed
a dramatically decreased viability (Fig. 1B). However, strikingly,
flies under identical experimental conditions (Fig. 1B). To further
confirm these results, we examined the survival rate upon infection
using another type of Gram-negative bacteria, Erwinia carotovora,
and obtained results similar to those of E. coli infection (Fig. 1B).
From these observations, we deduced that casparP1mutation
enhances resistance against Gram-negative bacterial infection.
caspar Mutation Induces Constitutive Expression of diptericin. Previ-
tightly correlates with induction of antibacterial peptides in the fat
elevates the expression of antibacterial peptide genes such as
diptericin. To test this hypothesis, we monitored diptericin gene
expression in uninfected and E. coli-infected flies by Northern blot
analyses. Interestingly, uninfected casparP1adults expressed dipteri-
level of diptericin expression was also observed in uninfected
on the PNAS web site). However, we found no expression of
diptericin in uninfected wild-type flies and heterozygous casparP1
mutants (Figs. 2A and 7).
To further confirm the constitutive expression of diptericin in
casparP1mutants, we used transgenic fly lines carrying a lacZ
reporter gene fused with the diptericin promoter (diptericin–lacZ)
(26). As previously reported (10, 27), the diptericin–lacZ reporter
activity was dramatically increased by E. coli infection (Fig. 2C).
Interestingly, consistent with our results obtained by Northern blot
analysis (Fig. 2A), casparP1mutants showed expression of the
diptericin–lacZ reporter even in the absence of bacterial infection
caspar Encodes a Drosophila Homolog of Mammalian FAF1. To study
the physical nature of the conceptual caspar gene, we performed
inverse PCR analysis using casparP1genomic DNA. Subsequently,
we found that casparP1flies contain an EP-element insertion at the
protein-coding sequence of the CG8400 gene (Fig. 1C) that may
hamper the gene expression of CG8400. Expectedly, although the
5? part of CG8400 transcripts was still present in the mutant (data
not shown), the 3? part of the transcripts downstream of the
Right. (B) The survival rate of w1118, key1, and casparP1adults at 1–7 days after E.
is significantly different from that of w1118at 7 days after infection (P ? 5.35 ?
10?4for E. coli infection, and P ? 6.16 ? 10?3for Er. carotovora infection). (C)
Schematic genomic organization of the caspar locus and transcripts. An EP-
element in the casparP1mutant (triangle) is inserted in the same direction as the
caspar gene at the 11,540,319th base pair of Drosophila melanogaster chromo-
some 2R sequence (release v. 4.3). (D) caspar expression in w1118, casparP1??
visualized by RT-PCR between exons 13 and 14. rp49 was used as a loading
(Casp), and human ETEA (hETEA). Drosophila CG10372 (CG10372), previously
inaccurately annotated as a fly FAF1, was revealed to be a Drosophila homolog
of ETEA proteins, constituting a protein family distinct from FAF1s. (F) caspar
expression at different stages of development. Northern blot analysis was per-
formed by using a 600-bp DNA fragment encompassing the central portion of
caspar coding region as probe. Ribosomal RNA (rRNA) was used as a loading
RT-PCR analyses of caspar were performed in various tissues. rp49 was used as a
Th, thorax; O, ovary; Te, testis.
Identification and characterization of caspar. (A) Ectopic melanization
and B) Northern blot quantifications of diptericin (dipt) (A) and drosomycin
(drom) (B) expression in w1118and casparP1adult flies at indicated times after
E. coli (A) and B. subtilis (B) infection. (C) Bright-field (BF) images of X-gal-
stained larval fat bodies from flies with indicated genotypes and treatments.
with indicated genotypes and treatments.
Mutation in caspar induces constitutive expression of diptericin. (A
Kim et al.PNAS ?
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EP-element insertion site was completely abolished by casparP1
mutation (Fig. 1D), demonstrating the absence of a full-length
transcription of CG8400 in the mutant. Moreover, casparP1?
Df(2R)ED2457 flies carrying a transallelic combination of casparP1
and a CG8400 deficiency allele displayed phenotypes identical to
those of casparP1homozygote mutants (Fig. 8, which is published
as supporting information on the PNAS web site). Furthermore,
expression of the CG8400 transgene fully suppressed diptericin
induction and melanization of uninfected casparP1mutants (Fig.
result from the loss of CG8400. Collectively, we concluded that
CG8400 and caspar essentially refer to the same gene and that
casparP1is a loss-of-function allele of CG8400. Therefore, we
named the uncharacterized gene CG8400 as caspar.
of human FAF1 (hFAF1), and all of the domains previously
identified in hFAF1 were conserved in Caspar (Fig. 10, which is
published as supporting information on the PNAS web site).
Furthermore, through phylogenic analyses of hFAF1 and related
of hFAF1 (Fig. 1E).
caspar Expression Is Abundant in the Fat Body. To examine the
temporal expression pattern of caspar during Drosophila develop-
ment, we performed Northern blot analysis using caspar-specific
probes. A single ?2.5-kb transcript was detected in all develop-
mental stages of Drosophila (Fig. 1F). Next, we dissected the larval
and adult tissues and subjected them to semiquantitative RT-PCR
experiments. Interestingly, caspar transcripts were highly enriched
in the fat bodies of larvae and adults (Fig. 1G). Because most
antibacterial proteins are expressed predominantly in this organ (1,
2), the abundance of caspar transcripts in the fat body properly
Caspar Suppresses the Immune Responses Induced by Bacterial Infec-
tion. To explore the molecular functions of Caspar, we generated
flies carrying the caspar transgene. The level of Caspar expression
varied among the transgenic alleles (Fig. 3A), allowing dose-
dependent studies of Caspar overexpression. Consistent with the
loss-of-function studies described above, overexpressed Caspar led
(Fig. 3B and Fig. 11, which is published as supporting information
on the PNAS web site). From these results, we deduced that
overexpression of Caspar may inhibit Drosophila immunity against
bacterial infection. To prove this hypothesis, we examined whether
Caspar overexpression would reduce the survival rate of infected
flies. Interestingly, fat body-specific expression of Caspar by yolk–
Gal4 and ubiquitous expression by da–Gal4 resulted in a decrease
in the viability of flies upon E. coli or Er. carotovora infection,
compared with flies overexpressing Imd or containing Gal4 alone
(Fig. 3 C and D, respectively). These results were similar to those
in key-null (key1) or relish-null (relishe20) flies, which are also highly
susceptible to bacterial infection (7, 12) (Fig. 3 C and D, respec-
fly extracts to have exponentially increased in Caspar-overexpress-
Gram-negative bacterial infection.
Caspar Inhibits Nuclear Localization of Relish. In the fat body, Relish
transcription factor migrates into the nucleus upon bacterial infec-
tion, and this nuclear translocation is critical for the infection-
dependent induction of antibacterial peptide genes such as dipteri-
cin (18, 19). Hence, we examined whether Caspar could suppress
diptericin induction by preventing the nuclear migration of Relish.
As previously reported (18), although Relish was predominantly
cytoplasmic in uninfected conditions, bacterial infection strongly
However, strikingly, overexpression of Caspar completely sup-
ing the protein in the cytoplasmic compartment (Fig. 4A). Con-
versely, fat body cells of casparP1flies exhibited ectopic nuclear
translocation of Relish even in uninfected conditions (Fig. 4B).
Therefore, we concluded that Caspar suppresses the nuclear local-
ization of Relish and the consequent antibacterial gene expression.
Caspar Is Not Involved in the Toll Pathway. Because Caspar inhibits
antibacterial immune responses, we were curious whether Caspar
could also inhibit the Toll pathway, an independent immune
pathway that regulates antifungal as well as anti-Gram-positive
bacterial immunity (1, 2). To address this idea, we examined the
expression levels of drosomycin, a representative target of the Toll
pathway, both in control and Caspar-overexpressing flies. As pre-
viously reported (5, 28), infection with Bacillus subtilis, Gram-
positive bacteria, strongly activated drosomycin gene expression
(Fig. 5A). Interestingly, overexpression of Caspar did not affect
drosomycin expression (Fig. 5A), and casparP1mutation also was
7). Moreover, the drosomycin–GFP reporter (10, 27, 29) was not
activated by casparP1mutation (Fig. 2D), although it was promi-
nently activated by bacterial infection (Fig. 2D). Furthermore,
Gal4-driven expression of Caspar was analyzed by immunoblot analyses using
anti-myc (Myc) and anti-tubulin (Tub) antibody. (B) Caspar suppresses diptericin
transcription in a dose-dependent manner. Adult female flies after E. coli infec-
rRNA was used as a loading control. (C and D) Survival rates of adult female flies
with indicated genotypes at 1–7 days after E. coli (C) and Er. carotovora (D)
infection. Bar graphs indicate survival rates at 7 days after infection (in C:*, P ?
viability of yolk?casp3or da?casp3flies was unaffected under uninfected con-
ditions (Fig. 9, which is published as supporting information on the PNAS web
site). (E) Bacterial colony counts were assayed from adult female flies at the
indicated times after infection with ampicillin-resistant E. coli. Representative
plates at 48 h after infection are shown. (F) Suppression of the casparP1pheno-
types by transgenic expression of Caspar. Adult female flies were analyzed for
melanization frequency (presented as a bar graph) and for diptericin expression
(Northern blot analysis in Inset). Genotypes of the flies are indicated.
Ectopic expression of Caspar impairs antibacterial immunity. (A) hs--
www.pnas.org?cgi?doi?10.1073?pnas.0603238103Kim et al.
overexpression of Caspar failed to suppress the elevated transcrip-
tion of drosomycin (Fig. 5B) as well as the ectopic melanization
phenotype of a Tl3mutant (Fig. 5C), whose Toll receptor contains
a dominant gain-of-function mutation (5, 30, 31). Therefore, we
concluded that Caspar does not affect the Toll pathway.
To further validate that Caspar is not involved in the Toll
migrates into the nucleus upon activation of the Toll signaling
of DIF was not affected by Caspar overexpression (Fig. 5D) or by
casparP1mutation (Fig. 5E), in stark contrast with the behavior of
Relish (Fig. 4).
Caspar Suppresses the Imd Pathway. It is well known that the Imd
pathway regulates Relish nuclear translocation and diptericin in-
and drosomycin expression (1, 2). Because Caspar specifically
inferred Caspar to be involved in the Imd pathway.
To substantiate the involvement of Caspar in the Imd pathway,
we performed genetic interaction assays using transgenic flies
expressing the components of the pathway. As previously reported
(6), overexpression of Imd robustly induced diptericin gene expres-
sion (Fig. 6A). However, coexpression of Caspar dramatically
inhibited the Imd-dependent induction of diptericin (Fig. 6A),
showing that Caspar is capable of suppressing the Imd-dependent
immune responses. Similarly, Caspar strongly suppressed diptericin
expression induced by other components in the Imd pathway
including FADD, TRAF2, and IKK? (Fig. 6A), implying that
Caspar-dependent inhibition of the Imd pathway occurs down-
stream of these molecules.
Caspar Inhibits the Dredd-Dependent Cleavage of Relish. At the end
point of the Imd pathway, Dredd-dependent cleavage of Relish is
essential for the nuclear localization of Relish (19). Therefore, we
suspected that Caspar suppresses the Dredd-dependent processing
of Relish to prevent its nuclear migration. To verify this possibility,
we examined Relish using immunoblot analyses. In uninfected
Relish was detected (18) (Fig. 6B). Upon bacterial infection, as
previously reported (18, 19), the 110-kDa band disappeared and
instead a 68-kDa band corresponding to a cleaved form of Relish
was newly detected (Fig. 6B). However, in the flies overexpressing
Caspar, the infection-dependent cleavage of Relish was completely
blocked (Fig. 6B). These results strongly suggested that Caspar
suppresses the nuclear translocation of Relish by inhibiting the
Interestingly, overexpression of Dredd alone has been reported
to be sufficient for inducing antibacterial gene transcription in the
absence of upstream infection signals (14) (Fig. 6C). To determine
whether Caspar is indeed able to suppress Dredd activity, we
examined diptericin expression in the flies coexpressing Dredd and
Caspar. Remarkably, diptericin expression induced by Dredd over-
expression was completely suppressed by Caspar (Fig. 6C). How-
induced by overexpression of the cleaved form of Relish (Fig. 6C),
which localizes to the nucleus and induces antibacterial gene
Imd pathway by blocking Dredd-dependent proteolytic activation
Finally, to investigate whether endogenous Caspar negatively
regulates the Imd pathway, we generated double-mutant lines
carrying both the casparP1mutation and a mutation affecting the
ingly, although the mutation of PGRP-LC, imd, or TAK1 did not
cells. Fat bodies isolated from adult female flies with the indicated genotypes
and treatments were analyzed by anti-N-terminal Relish immunostaining
(green, Anti-Rel). DNA was visualized by Hoechst 33258 (blue) in merged
images (Merge), showing the location of the nuclei. Fat droplets in these cells
generated a mesh-like appearance in the cytoplasm.
Caspar inhibits nuclear localization of Relish in Drosophila fat body
analyses of drosomycin expression in adult female flies with the indicated
conditions (B). rRNA was used as a loading control. (C) Melanization frequen-
cies (%) in adult female flies with indicated genotypes. Error bars indicate
standard deviations among three independent experiments. (D and E) Fat
bodies isolated from adult female flies with the indicated genotypes and
was visualized by Hoechst 33258 (blue) in merged images (Merge).
Caspar does not affect the Toll pathway. (A and B) Northern blot
Kim et al. PNAS ?
October 31, 2006 ?
vol. 103 ?
no. 44 ?
significantly affect diptericin gene induction caused by casparP1
mutation, the mutation of dredd or relish completely abolished it
(Fig. 6D), showing that endogenous Caspar controls the activity of
Dredd and Relish to suppress the Imd pathway. These genetic data
further confirmed our conclusion that Caspar specifically inhibits
Dredd-dependent cleavage of Relish (Fig. 6E).
Through a screen for Drosophila mutants with hyperactivated
Because mutation of caspar increased the resistance to infection by
in an antibacterial defense reaction that depends on the Imd
the antibacterial peptide diptericin even in uninfected conditions
(Fig. 2). Conversely, Caspar-overexpressing flies exhibited a strong
suppression of diptericin induction, highly increasing the flies’
susceptibility to bacterial infection (Fig. 3). These phenotypes were
strikingly similar to those observed in the loss-of-function mutants
for antibacterial immune signaling components (1, 2, 6, 17).
To dissect the molecular mechanism underlying the Caspar-
dependent inhibition of Drosophila immunity, we examined the
functional relationship between Caspar and the Imd pathway.
Surprisingly, Caspar specifically suppressed nuclear localization of
the Relish transcription factor (Fig. 4), and this suppression was
exerted by inhibition of the Dredd-dependent cleavage of Relish
(Fig. 6). These data also excluded the possibility that the diptericin
induction by casparP1mutation indirectly occurs by ectopic mela-
of Relish is regarded as a critical step in the Imd pathway, further
biochemical characterization of how Caspar inhibits the cleavage
of antibacterial signaling pathways in Drosophila.
Notably, Caspar contains multiple ubiquitin-related domains,
namely ubiquitin-associated (UAS) domain and ubiquitin-like do-
main, which are conserved in hFAF1 (Fig. 10). Because hFAF1
regulates protein degradation via these ubiquitin-related domains
(34), it is likely that Caspar performs similar biochemical activities.
Moreover, mutations of some components in the ubiquitin–
proteasome pathway, such as SkpA, dCullin, and Slimb (an F-box
protein), have been shown to suppress the Imd pathway by con-
trolling Relish activity (20). Therefore, it would be interesting to
investigate whether the ubiquitin-related domains of Caspar are
casparP1mutants also showed interesting semilethal phenotypes,
which might have resulted from deleterious side effects caused by
abnormally activated immune responses in uninfected and quies-
cent situations. However, because the lethality was only slightly
suppressed by a relish-null mutation (Fig. 12, which is published as
of the Imd pathway does not seem to be the sole cause of the
semilethality in casparP1mutants. Therefore, it would not be
surprising to find that Caspar may have additional physiological
roles that are independent of antibacterial immunity.
Because the loss of caspar is beneficial for efficient antibacterial
immune reactions (Fig. 1), Caspar down-regulation could actually
be advantageous for the survival of an infected organism. Intrigu-
ingly, we found decreased caspar transcript levels after bacterial
infection (Fig. 13, which is published as supporting information on
the PNAS web site), implying that, when the host is infected, the
activity of Caspar is suppressed in vivo. More importantly, the
initial infection when antibacterial immune responses had almost
organism to be more sensitive to immune defense; initial infection
induces a long-term silencing of caspar transcription to reinforce
antibacterial immunity against additional bacterial infection that
Because Caspar and the NF-?B-dependent immune signaling
pathways are highly conserved (1, 2), it is possible that the in vivo
functions of Caspar revealed in this study are conserved in mam-
regulation of the NF-?B pathway by FAF1 have been limited to
biochemical and cell culture-based experiments (23), our current
genetic studies using Drosophila will provide new insight into
understanding the in vivo functions of mammalian FAF1 as well as
negative regulation of mammalian innate immunity.
Fly Strains. Stocks were raised on a standard cornmeal–yeast agar
medium at 25°C. Each EP line carries a P-element containing
Gal4-binding sites and a basal promoter oriented to direct expres-
For the screening, we scored homozygous phenotypes of 15,000
independent nonlethal EP lines from the GenExel’s EP fly collec-
tion without using any Gal4 drivers. w1118was used as wild type
because it is the parental line of all of the GenExel EP lines.
UAS-Imd, EP(X)DTRAF2, UAS-IKK?, UAS–dredd, UAS–relish,
Tl3, PGRP–LC?E, dreddB118, imd1, TAK11, key1, relE20, diptericin–
lacZ, drosomycin–GFP, hs–Gal4, da–Gal4, and yolk–Gal4 fly lines
are described elsewhere (5–7, 10–12, 14, 17, 26, 29, 37).
EP(3)FADD was purchased from GenExel. Transgenes were ex-
pressed by using the Gal4?UAS binary system. To generate UAS–
caspar flies, caspar EST cDNA (Berkeley Drosophila Genome
adult female flies using anti-N-terminal Relish antibody. The flies were in-
fected (?) or kept uninfected (?) at 4 h before sampling. Asterisks indicate
nonspecific bands. Transgenic Caspar was detected by anti-myc antibody.
Tubulin was used as a loading control. (C) Uninfected adult female flies with
indicated genotypes were subjected to Northern blot analyses of diptericin.
adult flies with indicated genotypes after E. coli infection (?) or no infection
(?). (E) A schematic model of in vivo roles of Caspar in Drosophila.
Caspar suppresses the Imd pathway by inhibiting Dredd-dependent
www.pnas.org?cgi?doi?10.1073?pnas.0603238103 Kim et al.
Project accession no. LD03368) was cloned into the myc-tagged
pUAST vector and microinjected into w1118embryos.
Infection Experiments. Bacterial infection was carried out by prick-
ing third-instar larvae or 7-day-old adults with a thin needle dipped
into a concentrated solution of the following bacterial strains:
wild-type E. coli, ampicillin-resistant E. coli, Er. carotovora, or B.
subtilis. Survival experiments were performed with 50 flies at 25°C
for each fly line grown in the same conditions. Surviving flies were
transferred to fresh vials, and counts were taken every day for 7
days. For Figs. 1 B and 2 C and D, the average of three replicate
experiments is presented as a graph, and the standard deviation is
indicated as an error bar (5, 16). P values were calculated by
one-way ANOVA. Examination of bacterial growth in flies was
performed as previously described (5). The average of three
replicate experiments is presented as a bar graph, and the standard
deviation is indicated by an error bar.
Histology and Molecular Analyses.Immunostainingoffatbodieswas
carried out as previously described (10) by using anti-N-terminal
Relish (18) or anti-DIF (32) antibody. X-gal staining of fat bodies
was performed as previously described (10). Immunoblot analysis
was performed as previously described (38) by using anti-N-
terminal Relish antibody. RT-PCR analysis was performed as
previously described (38). Primer sequences for RT-PCR of caspar
mRNA are 5?-GACGCGAGTCCATCAGATTAG-3? (3? region,
forward), 5?-CAGCTTGAGCGACTCCAATG-3? (3? region, re-
verse), 5?-ATGTCAGAGAACAAGGACGAG-3? (5? region, for-
ward), and 5?-GATGTGCGGCTGATTTAGGTTTAG-3? (5? re-
gion, reverse). Northern blot experiments and quantification of the
signals on three independent blots were conducted as previously
described (5). More specifically, signals of diptericin or drosomycin
expression were normalized with a corresponding value of rRNA
signals. The levels of diptericin in wild-type flies 6 h after infection
and the levels of drosomycin in wild-type flies 12 h after infection
were each standardized as 100, and the results are presented as
relative activity in percent, in two separate graphs. For Fig. 2 A and
B, the averages of three independent experiments are presented as
Melanization frequencies were calculated in proportion of the
number of flies with melanization mass to the total number of flies
(n ? 100) from three independent experiments under the same
conditions. For Fig. 5C, the averages of three independent exper-
indicated by error bars.
We thank Drs. B. Lemaitre (CNRS), Y. Engstrom (Stockholm Univer-
sity), J. Royet (Universite Louis Pasteur), W. J. Lee (Ewha Woman’s
University), D. Hultmark (Umeå University), K. Anderson (Sloan–
Kettering Institute), M. Meister (Universite Paul Sabatier), S. Stoven
(Umeå University), and the Bloomington Stock Center for providing fly
stocks and antibodies.
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