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: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
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vol. 103 ?
no. 44 www.pnas.org?cgi?doi?10.1073?pnas.0603238103
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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