REL1, A HOMOLOGUE OF DROSOPHILA DORSAL, REGULATES TOLL ANTIFUNGAL IMMUNE ...

17 Pages·2005·0.79 MB·English

JBC Papers in Press. Published on February 18, 2005 as Manuscript M500711200
REL1, A HOMOLOGUE OF DROSOPHILA DORSAL, REGULATES TOLL ANTIFUNGAL
IMMUNE PATHWAY IN THE FEMALE MOSQUITO AEDES AEGYPTI.
Sang Woon Shin, Vladimir Kokoza, Guowu Bian, Hyang-Mi Cheon, Yu Jung Kim
and Alexander S. Raikhel*
From the Department of Entomology and the Institute for Integrative Genome Biology, University
of California, Riverside California 92521, USA
Running Title : Aedes REL1 in mosquito immunity
Address correspondence to : Alexander S. Raikhel, Department of Entomology and the Institute for
Integrative Genome Biology, UCR, Riverside, California 92521, Tel: [phone]; FAX: 909-787-
2130; e-mail: [email protected]
Signaling by Drosophila Toll pathway Mosquitoes transmit many diseases,
activates two Rel/NF-κB transcription factors— including malaria, which is a particularly
Dorsal (Dl) and Dorsal-related immune factor threatening disease that is responsible for over two
(Dif). Dl plays a central role in the million deaths per year (1, 2). Diseases caused by
establishment of dorso-ventral polarity during mosquito-borne viruses, most importantly Dengue
early embryogenesis, whereas Dif mediates the fever, are reaching disastrous levels in Central and D
o
w
Toll receptor-dependent antifungal immune South America and Southeast Asia (3). Since n
lo
response in adult Drosophila. The absence of a arriving on the eastern coast of the U.S. in 1999, ad
e
DDilf moratyh oplloagy iints mfuonsqctuioitnoa gl ernoolem iens tshueg gmesotssq tuhitaot twhees twWaerdst, kNilillien g eanlcmeopshta lait ish unvdirruesd pheaosp les w(e4p)t. d from
Toll-mediated innate immune responses. We Lymphatic filariasis, a nematode-based disease http
have cloned and molecularly characterized the transmitted by mosquitoes, affects millions of ://w
w
gene homologous to Drosophila Dl and to people in tropical regions of the world (5). The w
Anopheles gambiae REL1 (Gambif1) from the major reasons for this tragic situation are the .jbc
.o
yellow fever mosquito Aedes aegypti, named unavailability of effective vaccines for malaria and rg
AaREL1. AaREL1 alternative transcripts other mosquito-borne diseases and the lack of by/
g
encode two isoforms, AaREL1-A and AaREL1- insecticides and drug resistance to vectors and u
e
s
B. Both transcripts are enriched during pathogens, respectively (6, 7). t o
n
embryogenesis and are inducible by septic Immunity plays a key role in the interaction N
o
injury in larval and female mosquitoes. between a pathogen and its vector host. Many ve
m
AaREL1 and AaREL2 (Aedes Relish) genes and their products involved in mosquito be
selectively bind to different κB motifs from innate immunity have been identified and r 18
insect immune gene promoters. Ectopic characterized either independently or on the basis , 20
1
8
expression of AaREL1-A in both Drosophila of their relationships to known immune genes of
mbn-2 cells and transgenic flies specifically the model organism, Drosophila melanogaster.
activates Drosomycin and results in increased The genomic DNA sequences of Anopheles
resistance against the fungus Beauveria gambiae, a major disease vector involved in the
bassiana. AaREL1-B acted cooperatively with transmission of Plasmodium falciparum malaria in
AaREL1-A to enhance the immune gene Africa, have been revealed, and they encompass
activation in Aag-2 cells. The RNAi knockouts more than 278 million base pairs, which suggests
revealed that AaREL1 affected the expression the presence of ~14,000 protein-encoding genes
of Aedes homologue of Drosophila Serpin-27A (8). Of these, 242 An. gambiae genes from 18 gene
and mediated specific antifungal immune families have been implicated in innate immunity
response against B. bassiana. These results by comparative genomic analysis to the
indicate that the homologue of Dl, but not that Drosophila immune systems (9). The mosquito
of Dif, is a key regulator of the Toll antifungal immune genes involved in recognition, signal
immune pathway in Ae. aegypti female modulation, and effector systems diverge widely
mosquitoes. from those in Drosophila. In addition, these
families of immune factors have undergone
1
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
significant expansion during the evolution of regulator in determining dorso-ventral polarity
mosquitoes, which possibly reflects different (18). A gradient of nuclear Dl spatially restricts
selection pressures to a variety of pathogens the expression of zygotic genes along the dorso-
encountered in these insects’ distinct lifestyles. ventral axis and functions both as a transcriptional
However, the components of two principal activator and a repressor (19). The remarkable
immune transduction pathways, the Toll and IMD, structural and functional similarities between the
have principally been conserved between these mechanisms of activation of Dl during Drosophila
two insects. This points to the evolutionary morphogenesis and NF-κB during the mammalian
requirement of preserving the integrity of key acute phase response have implicated Dl and Dif
factors in intracellular immune signal transduction in the host defense of Drosophila (20, 21).
and gene activation pathways (9). However, two independent studies clearly show
In Drosophila, three Rel/NF-κB molecules— that Dif, the possible duplicator of the Dl gene
Dorsal-related immunity factor (Dif), Dorsal (Dl), during Drosophila evolution, is the essential
and Relish—affect the expression of numerous regulator of the Drosophila immune Toll pathway.
immune genes, including those encoding anti- The rescue experiments of Drosophila Dl and Dif
microbial peptides (AMPs) (for review, see 10, double mutants have shown that the ectopic
11). These Rel/NF-κB molecules are involved in expression of Dif without Dl is sufficient to
D
two distinct innate immune pathways: the Toll mediate the induction of Drosomycin (22). In ow
n
pathway, which is mediated by Dl and Dif and addition, genetic epistasis studies with Dif mutant lo
a
d
responds primarily to fungal and Gram-positive flies have demonstrated that Dif mediates the Toll- e
d
bacterial infections, and the IMD pathway, which dependent control of the inducibility of the fro
m
is regulated by Relish and is predominantly Drosomycin gene (23). h
dTiorellc-tseigdn aalginagin st paGthrawma-yn, egaDtiivfe bsaticmteurliaat. esI n tthhee aspecTtsh eo fD trhoes oipmhmilau nTeo lrle pspaothnwsea y( froerg urelavtieesw m, asneye ttp://ww
w
production of the antifungal factor Drosomycin 24). In addition to activating antifungal defenses, .jb
c
(12). In loss-of-function mutants affecting the Toll this pathway is also required for resistance against .o
rg
signaling pathway, the inducibility of Drosomycin Gram (+) bacteria, for regulation of the b/
y
in immune-challenged adults is severely melanization cascade and blood cell proliferation. g
u
compromised, whereas those of Defensin, Drosophila mutants that constitutively activate es
Cecropin, and Attacin are reduced to a lesser Toll pathway, such as necrotic, Toll10b, and cactus, t on N
extent. In contrast, the expression of Diptericin, all show hemocyte phenotype with over-reactive ov
e
Drosocin, and Metchnikowin are completely blood cells that form melanotic masses (25), mb
e
unaffected (13, 14). In Drosophila relish mutants, similar to the cellular response to parasites (26). r 1
8
the induction of immune defense is severely Surprisingly, the An. gambiae genome , 2
0
reduced, and insects become extremely sensitive to sequence harbors only two NF-κB genes, REL1 18
bacterial and fungal infection (15). Immune (gambif1) and REL2, which are homologues of
effector genes under the regulation of Relish Drosophila Dl and Relish, respectively (9, 27).
include Cecropin, Diptericin, Attacin, Defensin, The An. gambiae Dl homologue Gambif1 (now
and Metchnikowin (15, 16). Recent micro-array called REL1) can bind to κB motifs on the
studies have shown that in addition to AMP genes promoters of Drosophila Diptericin and Cecropin
many other immune genes are regulated and activate transcription of a reporter gene under
cooperatively or independently by the Toll and the control of the Diptericin κB motif in cell
Imd pathways (17). These genes include culture studies (28). The lack of a Dif ortholog in
recognition molecules like PGRPs and GNBP, mosquitoes raises a question about the difference
which are components involved in both the Imd between their immune responses and those of
and Toll pathways; the protease cascade proteins; adult Drosophila and about the immune function
and putative components of the phenoloxidase or of mosquito homologue of Dl (REL1). In
blood clotting pathways. particular, it has not been demonstrated in vivo
Dl, the first insect member of NF-κB family, whether mosquito REL1 serves as a functional
was identified in a screen for genes required for DIF analog in adult mosquitoes. In this report, we
Drosophila embryonic development. Dl is a key have cloned and characterized a homologue to
2
Drosophila Dl from the yellow fever mosquito 2 days after blood feeding. The unfertilized eggs
Aedes aegypti, which we named AaREL1. In vitro were obtained by dissecting ovaries from
comparative binding studies, transfection assays in mosquitoes 3 days after blood feeding, while
both Drosophila mbn-2 cells and mosquito Ae. fertilized eggs were collected 1 day after egg
aegypti Aag2 cells, the transgenic over-expression laying. Total RNA was prepared by the Trizol
in flies, and in vivo studies by the RNAi knockouts technique (GIBCO/BRL). Samples of 5 µg total
in mosquitoes provide conclusive evidence to RNA were separated on a formaldehyde gel,
implicate AaREL1 as the key activator of the Toll- blotted, and hybridized with a corresponding DNA
mediated antifungal immune pathway in adult probe. RT-PCR was performed by using the Titan
female mosquitoes. one-step RT-PCR kit (Roche) with samples of 0.2
µg total RNA as templates. Tubes containing RNA
EXPERIMENTAL PROCEDURES and RNase inhibitor (1 u/µl, Roche) were
incubated for 30 min at 50 °C for RT-reaction.
Isolation of cDNA and genomic DNA Clones and Amplification conditions included rapid heating to
RACE—A PCR product was obtained from 94 °C for 2 min followed by 25 to 30 cycles of
genomic DNA by degenerate primers based on the 55 °C for 1 min, 72 °C for 3 min, and 94 °C for 45
conserved region of the RHD from Drosophila Dl s.
D
and Anopheles REL1 (gambif1). The following Electrophoretic gel mobility shift assay (EMSA)— o
w
n
primers were used: AaREL1-R2 (5’- Each protein was synthesized by a coupled in vitro lo
a
CAGTG(T/C)GCCAA(A/G)AAGAAGGA-3’) transcription-translation (TNT) system (Promega). de
d
and AaREL1-R-A (5’- The corresponding cDNA clones were subcloned fro
m
GGCAATCTTCTCGCACAGCA-3’). This PCR into pcDNA3.1/Zeo (+) (Invitrogen). The in vitro h
fragment was subcloned using a TA cloning kit transcription-translation reactions programmed by ttp
(Novagen) and was then utilized as a probe to the circular plasmid DNA utilized the T7 promoter. ://w
w
w
screen the Lambda ZAPII cDNA library prepared To confirm the synthesis of proteins with expected .jb
from previtellogenic female Ae. aegypti size, the control TNT reactions of each protein c.o
mosquitoes. Six clones were isolated and were performed in the presence of [35S] brg/
y
sequenced from both the 5’ and 3’ ends. Based on methionine, and the resulting reactions were g
u
sequencing and restriction mapping analyses, the analyzed by SDS-polyacrylamide gel es
t o
six clones were subdivided into two groups. The electrophoresis (PAGE) and autoradiography. The n
N
longest representative of each cDNA group, the annealed deoxyoligonucleotide of µB motifs were o
v
e
D4 and D6 cDNA clones, were fully sequenced purified from 15 % TBE Criterion Precast Gel m
b
e
from both strands. The 5’ and 3’ ends of D4 and (Bio-Rad) and labeling of double-stranded r 1
8
D6 cDNA clones were identified by the RACE oligonucleotides and EMSA was performed with a , 2
0
system (GIBCO/BRL). Two overlapping PCR gel shift assay system (Promega). The protein- 18
products spanning 13,355 bp in length were DNA complex was separated on 5% TBE
isolated using the Expand Long Template PCR Criterion Precast Gel (Bio-Rad) and visualized by
system (Roche). These PCR fragments were auto-radiography.
subcloned using a TOPO XL cloning kit Transfection assay in Drosophila mbn-2 cell
(Invitrogen) and then sequenced. The sequences line—Coding region sequences of Ae. aegypti—
reported in this paper have been deposited in the ∆REL2 (Relish C8 cDNA, which encodes
GenBank database (accession nos. AY748242 for truncated Rel-type protein without N-terminal
AaREL1-A cDNA, AY748243 for AaREL1-B transactivation domain, 29); AaREL2+ (Rel-type
cDNA, and AY748244 for genomic DNA) cDNA with N-terminal transactivation domain of
Northern hybridization and RT-PCR—Adult 2- or Aedes REL2, 29); AaREL1-A (D4 cDNA); and
3-day-old Ae. aegypti females were injected with a AaREL1-B (a RT-PCR product with ORF of 844
stationary phase culture of Enterobacter cloacae amino acids)—were PCR amplified and inserted
and/or Micrococcus cloacae. For the stage-specific into pMT/V5-His (Invitrogen). They were used
study, adult males, females and fourth instar larvae for making transformation vectors for each NF-κB
were collected with or without bacterial challenge. protein. Drosophila mbn-2 cells were seeded at 2
The vitellogenic mosquitoes were collected 1 and x 106 cells per ml in 35 mm plates of Drosophila
3
Schneider’s medium (GIBCO) supplemented with reactions in a 1.0% agarose gel in TBE (90 mM
5% fetal bovine serum (GIBCO) and 1X Gluta Tris-borate/2 mM EDTA, pH 8.0). A Picospritzer
Max-1 (GIBCO). Twenty-four hours later, the II (General Valve, Fairfield, NJ) was used to
cells were co-transfected with 2 µg of expression introduce 200 nl of dsRNA into the thorax of CO -
2
plasmid. The cells were incubated for 4 h in anesthetized mosquito females, at 2–3 days post-
serum-free Schneider’s medium, which was then eclosion.
removed and replaced with complete medium. Septic injury and survival experiments—Septic
Over-expression was induced by 48 h of injuries were performed by pricking the flies or
incubation with the addition of 500 µM CuSO . mosquitoes in the rear part of the abdomen with a
4
Immune response was activated by the addition of Hamilton 31S needle dipped into bacterial culture
heat-inactivated E. coli for 4 h. or a fungal spore suspension. Survival experiments
Transfection assay in Aag-2 cell line—Cell line were carried out under the same conditions.
Aag-2 from Ae. aegypti was maintained in Groups of 20 female Drosophila or Ae. aegypti
Schneider medium (Invitrogen) supplemented with female mosquitoes after 2 d recovery following
10% fetal bovine serum (FBS, Hyclone). Aedes AaREL1 dsRNA injection were challenged by
REL2+, AaREL1-A, and AaREL1-B were inserted bacteria culture or a spore suspension (about 5 x
into the pAc5.1/V5/HisA (Invitrogen) vector. 107 viable spores / ml) of B. bassiana strain GHA.
D
Cells were incubated at 26 oC for 36 h prior to The viable spore number was calculated by o
w
n
transfection. After transfection, the cells were spreading the suspension onto Sabouraud dextrose lo
a
incubated at 26 oC until they reached at least 70% agar plates. de
d
confluency (approximately 24 h). Following fro
m
transfection, for the bacterial challenge, heat- RESULTS h
inactivated E. coli was added to the well and ttp
incubated for an additional 4 h, then cells were Cloning of two REL1 isoform cDNAs from Ae. ://ww
w
harvested for RNA extraction. aegypti—A 376-bp DNA fragment was obtained .jb
Generation of transgenic flies—AaREL1-A cDNA using a set of sense and anti-sense primers c.o
(D4) was subcloned downstream of the UAS (AaREL1-R2 and AaREL1-R-A) and was used as brg/
y
sequence in piggyBac 3xP3-EGFP-based vector. a probe to screen the cDNA library prepared from g
u
Transformants were established by micro-injection previtellogenic Ae. aegypti female mosquitoes. es
t o
of wild-type Canton-S strain homozygous for The nucleotide sequence of the longest 2,250-bp n
N
white mutation. Two independent Drosophila lines D4 clone out of a total of 6 clones contained a full o
v
e
were examined, and both produced similar results open reading frame that was conceptually m
b
e
in over-expression of AaREL1-A transgene driven translated into a 579 amino acid-long polypeptide. r 1
8
by Hsp70-GAL4 control element-using driver line Additional 5’-untranslated region (UTR, 41-bp) , 2
0
P{GAL4-Hsp70.PB}2, which was kindly provided and 3’-UTR (11-bp) sequences that were followed 18
by the Indiana University Drosophila stock center by poly (A) sequence were identified by RACE-
(Bloomington, IN) PCR. The sequence and overall structure of the
Synthesis and micro-injection of dsRNA— conceptual protein from D4 was highly similar to
Synthesis of dsRNA was accomplished by Drosophila Dl and An. gambiae REL1 (AgREL1),
simultaneous transcription of both strands of and thus the Ae. aegypti homologue was named
template DNA with a HiScribe RNAi AaREL1-A (Fig. 1A). A phylogenetic analysis
Transcription Kit (NEB). The plasmid LITMUS using Rel-homology domains demonstrated that
28iMal containing a nonfunctional portion of the E. AaREL1 and AgREL1 clustered with Drosophila
coli malE gene that encodes maltose-binding Dl. This Dl /REL1 subgroup, together with
protein was used to generate control dsRNA. After Drosophila Dif, formed a separate cluster that was
RNA synthesis, the samples were treated by different from the group of mammalian Rel
phenol/chloroform extraction and ethanol proteins (Fig. 1B).
precipitation. The dsRNA was then suspended in Sequencing of another cDNA clone, D6,
diethyl pyrocarbonate-treated distilled water with indicated differences in the 5’-UTR sequence that
a final concentration of 5 µg/µl. The formation of contained an alternative start codon. Both D4 and
dsRNA was confirmed by running 0.2 µl of these D6 cDNA clones shared identical sequences only
4
in the RHD region, but the C-terminal domains residues, KRKKRK, whereas the monopartite-like
(CTDs), including the nucleus localization signal sequence of AaREL1-B was SRKKSK and
(NLS), were completely divergent. An additional included an upstream cluster of basic residues,
5’-UTR sequence of 263 bp of D6 cDNA was KNKK.
obtained by 5’-RACE PCR. Two major PCR Two overlapping PCR products spanning
products, short and long form, obtained by 3’- 13,355 bp in length were isolated and sequenced.
RACE-PCR, added the 3’-truncated region of the This genomic region did not contain a 5’-UTR, N-
D6 cDNA. Both short and long RT-PCR products terminal region (M1-C150), and short 3’-UTR as
were subcloned and additional transcript shown in the D4-type transcript. The sequence
sequences of 1,290 bp and 1,967 bp were obtained. result, however, gave us sufficient information
The complete short- and long-form transcripts about how the RNA was processed in generating
were 3,433 bp and 4,110 bp in size, which the different types of AaREL1 transcripts (Fig. 1C).
encoded the same protein. The additional 3’-UTR These exon-intron structures and the alternative
of 4,110-bp transcript was identical to the 3’- splicing of the AaREL1 gene were very similar to
region of D4 cDNA clone, indicating that long- the Drosophila Dl gene (30), showing that both
form transcript might be the precursor mRNA of insect genes originated from a common ancestor
the D4-type transcript. The 844-residue protein gene.
D
encoded by these transcripts lacked N-terminal 39 o
w
n
residues that were encoded by the AaREL1-A The inducible AaREL1 isoform transcripts are lo
a
transcript, which indicated the presence of an expressed in adult Ae. aegypti females—Northern de
d
alternative transcription start site. We were able to blot analyses were performed to examine the fro
m
isolate 2,894-bp (844 residues) and 2,968-bp (883 expression of AaREL1 transcripts. Utilization of h
residues) cDNA fragments by RT-PCR that the total RNA revealed three transcript bands of ttp
yielded polypeptides with or without N-terminal ~2.5 kb, ~3.5 kb, and ~4.3 kb in size that were ://w
w
w
39 residues. Northern blot analysis clearly showed constitutively expressed during the normal .jb
that all Ae. agypti REL1 transcripts used both start unchallenged state of the naïve larvae in adult c.o
sites, which resulted in doublet bands for each males and females (Fig. 2A). Probe from brg/
y
alternate transcript (Fig. 2A). AaREL1-B-specific CTD region (F400-L568 of g
u
The CTD of the second Ae. aegypti REL1 AaREL1-B) did not hybridize to the ~2.5-kb band, es
t o
form (named AaREL1-B) was highly conserved suggesting that it represented AaREL1-A transcript n
N
with the rear portion of Drosophila Dl-B CTD, (Fig. 2A). According to the sizes of the transcripts, o
v
e
which possessed trans-activating properties (Fig. the ~3.5-kb and ~4.3-kb bands corresponded to the m
b
e
1A). A crucial region for the transactivation short and long AaREL1-B transcripts, respectively. r 1
8
properties of Drosophila Dl-B, located between The expression level of all transcripts in larvae and , 2
0
amino acids 576 and 683, contains two putative adult female mosquitoes were more elevated 5 h 18
acidic activation modules (30). These two modules after septic injury with a mixture of stationary-
and a bipartitate NLS were highly conserved in the phase culture of Enterobacter cloacae and
CTDs of Drosophila and AaREL1-B. A Q-rich Micrococcus luteus. AaREL1 transcripts remained
region, present in the CTD of Drosophila Dl-B, unchanged upon bacterial challenge in adult males.
was absent in AaREL1-B. The Rel domains of Dl- Without immune challenge, the expression
B and AaREL1-B lacked an NLS at their ends. level of AaREL1 transcripts was very low in the
Instead, both Dl-B and AaREL1-B had a bipartite fat body and mainly occurred in the ovary of naïve
NLS in their CTDs. This was a common variant pre-vitellogenic female mosquitoes. The AaREL1
for NLS in which a small cluster of basic residues gene expression was elevated after blood feeding
positioned 10–12 residues at the N-terminal of a as well as after bacterial challenge, and it
monopartite-like NLS sequence. The additional increased further during egg development. All
binding energy contributed by the upstream cluster REL1 transcripts showed the highest level in
of basic residues relaxes the requirements for the unfertilized eggs that were dissected from ovary of
downstream monopartite-like sequences (31). As female mosquitoes 3 d post-blood feeding (PBF)
shown in Figure 1A, the monopartite NLS and in 1-d post-oviposition eggs (Fig. 2A). These
sequence of AaREL1-A consisted of all basic AaREL1 expression data in the ovary and during
5
vitellogenesis, and in eggs of Ae. aegypti, specific oligonucleotide (I) effectively completed
indicated REL1 participation in the Toll-mediated binding to the labeled probe, whereas the addition
early embryogenesis similar to that of Drosophila of a nonspecific competitor, AP2, affected binding
Dl. In addition, AaREL1 expression was greatly very weakly (Fig. 2C). The competition of
induced after septic injury in the fat body of pre- AaREL1-A binding to I motif by three κB motifs
vitellogenic female mosquitoes, which implicated (V, VI, and VII) was similar to that of the
AaREL1 involvement in innate immunity at this nonspecific competitor, indicating that binding of
stage of the mosquito life cycle (Fig. 2A). AaREL1-A to these motifs was nonspecific (Fig.
2D). The binding specificities of AaREL1-A and
Comparative analysis of Ae. aegypti REL1 AaREL2 to various κB motifs from insect immune
isoforms and REL2 (Relish) binding to κB gene promoters were clearly different (Fig. 2B).
motifs—We expressed AaREL1-A and AaREL1-B To test whether AaREL1-A and AaREL2 could
by using an in vitro coupled transcription- bind to κB motifs as heterodimers, equal amounts
translation assay. We used D4 cDNA for of both these Rel proteins, co-translated in vitro
AaREL1-A and a 2,894-bp RT-PCR product (data not shown), were subjected to EMSA.
containing the alternative ORF of 844 residues for Formation of additional complexes was not
AaREL1-B. In vitro translation of D4 cDNA detected, indicating that AaREL1-A and AaREL2
D
resulted in two REL1-A protein bands, indicating bound to tested κB motifs only as homodimers ow
n
that an alternative start site could be used as the (Fig. 2A). Binding activity of in vitro translated lo
a
d
translation initiation codon (data not shown). Only AaREL1-B was not detected with any κB motif ed
a single major band was observed after in vitro (data not shown). In addition, its co-translation fro
m
translation of the REL1-B RT-PCR product. The with either AaREL1-A or AaREL2 did not affect h
sizes of translated proteins matched well with κB binding of these two factors. ttp://w
those expected from deduced proteins (data not w
w
shown). For in vitro translation of AaREL2, we AaREL1-A activates Drosomycin expression in .jb
c
used our previous construct (29). Seven κB motifs, Drosophila mbn-2 cell line and transgenic flies— .org
including those from Drosophila Cecropin A1 and Drosophila tumorous blood cells (mbn-2 line) can b/
y
Ae. aegypti Defensin promoters (29), were be induced to express Diptericin and Cecropin gu
e
designed based on the comparison of promoter s
genes by the addition of lipopolysaccharide (LPS) t o
sequences of various insect immune genes (Fig. to the culture medium (33, 34). Moreover, n N
2B). expression of the Drosomycin gene is highly ove
m
In EMSA, in vitro expressed Dl-A activated by immune challenge in this cell line b
e
specifically bound to four κB motifs among seven (35). We examined the expression of Drosophila r 1
8
tested motifs (Fig. 2B). The highest affinity of AMP genes in the presence of exogenous , 20
1
AaREL1-A binding was to the κB motif (I) from mosquito NF-κB proteins. The over-expression of 8
Drosophila CG16978 promoter. This κB motif, 5’- mosquito AaREL1-A in mbn-2 cells activated
GGGAAATTCC-3’, from the promoter of strong expression of the Drosophila Drosomycin
Drosophila unknown immune gene CG16978, gene in the absence of bacterial challenge (Fig.
matched well to κB motif consensus, 3A). The expression pattern of Diptericin,
GGG(W)nCCM, of Drosophila zen Ventral Cecropin, and Attacin genes remained constant
Repression Element (32). This type of κB motif during over-expression of any mosquito Rel
was present in the promoter regions of Drosophila protein (Fig. 3A, data not shown).
immune genes whose expression was activated Next, the mutant flies, which over-expressed
only by the Toll pathway (17). Similar κB motifs AaREL1-A by the heat shock (hs)-GAL4/UAS
were found from Drosophila GNBP-like system, were constructed to show in vivo activity
(CG13422) and Nec gene promoters (Table. 1). of AaREL1-A. Due to the leaky promoter,
The other six sites were representatives of various AaREL1-A had a background expression which
κB motifs found in upstream promoter regions of could activate the Drosomycin gene independent
Drosophila and mosquito immune genes (Fig. 2B). of septic injury with fungal infection (Fig. 3B).
The addition of 25-fold excess of the unlabeled The over-expression of AaREL1-A by heat shock
6
resulted in an increased expression level of the response of Ae. aegypti REL1 and Serpin-27A was
Drosomycin gene but not other Drosophila AMP elicited only by the fungal challenge (Fig. 4B).
genes such as Diptericin, Cecropin, and Attacin Next, dsRNA complementary to the RHD of
(data not shown). The level of Drosomycin AaREL1 was synthesized in vitro and injected into
expression is significantly higher in heat-shocked the thorax of newly emerged female mosquitoes.
flies over-expressing AaREL1-A than that fully When AaREL1 dsRNA was introduced into the
induced by immune challenge in the presence or mosquitoes, the mRNA level of both AaREL1
absence of bacterial challenge. (Fig 3B). These transcripts greatly decreased (Fig. 4C). The
transgenic flies exhibited increased resistance to mRNA quantity of Ae. aegypti Serpin-27A in
the infection with B. bassiana spores (Fig. 3C). AaREL1 dsRNA-treated mosquitoes also
significantly declined, suggesting that AaREL1
REL1-A and REL1-B synergistically activate the was involved in the regulation of Ae. aegypti
immune genes in Aag-2 mosquito cells—Aag-2 Serpin-27A gene expression. The mRNA level of
cell line originated from Ae. aegypti embryonic AaREL2 was not affected by the treatment of
tissues is responsive to the immune challenges AaREL1 dsRNA.
(36). Immune factors such as cecropin, defensin, Genetic analyses have shown that Drosophila
lysozyme, and transferrin were inducibly Toll pathway mutants are sensitive to fungal
D
expressed in this cell line. Drosophila Serpin-27A, infection (13). To address the role of Aedes Dorsal o
w
n
which regulates the melanization cascade through homologue in mosquito immune response, the lo
a
the specific inhibition of the PPAE, is an acute susceptibility of either AaREL1 or AaREL2 de
d
immune-responsive gene mainly regulated by the dsRNA-treated mosquitoes was compared after fro
m
Toll pathway (37). We have isolated the mosquito bacterial or fungal challenge. After 2–4 d recovery h
homologues of Drosophila Serpin-27A from Ae. following injection with either dsRNA, the treated ttp
aegypti (Shin, S.W., Antonova, Y., and Raikhel, mosquitoes were challenged by either the spores ://w
w
w
A.S., unpublished data). In addition to Aedes of entomopathogenic fungus, B. bassiana, or the .jb
Cecropin A and Defensin A, the expression of mixture of E. cloacae and M. luteus. As illustrated c.o
mosquito Serpin-27A were induced by heat-killed in Figure 4D, the AaREL1 dsRNA-treated brg/
y
bacteria in Aag-2 cells (Fig. 4A). mosquitoes were significantly more sensitive to g
u
Transfection of AaREL1-A alone in Aag-2 the fungal infection than AaREL2 or MalE es
t o
cells could weakly activate mosquito immune dsRNA-treated mosquitoes. In contrast, only n
N
genes. However, when both AaREL1-A and AaREL2 dsRNA-treated mosquitoes were o
v
e
AaREL1-B were co-transfected together, the same susceptible to the bacterial challenge (Fig. 4E). m
b
e
immune genes were strongly activated (Fig. 4A). We did not observe any increased susceptibility to r 1
8
This experiment showed that AaREL1-B acted Gram-positive bacteria in either AaREL1 or , 2
0
cooperatively with AaREL1-A in enhancing the AaREL2 dsRNA-treated mosquitoes (data not 18
immune gene activation. This response was shown).
similar to that observed for Drosophila Dl-B,
which increased in vitro activation of κB-like DISCUSSION
promoters when it was co-expressed with Dl-A
(30). Because AaREL1-B did not bind to any κB We have investigated the contribution of
motifs directly, it might act as a co-factor to REL1 in the antifungal immune defense in adult
AaREL1-A. females of Ae. aegypti mosquitoes. This
investigation has been prompted by the lack of
Aedes REL1 regulates antifungal immune homologue of Drosophila Dif in mosquitoes (9).
response in vivo—We tested the expression Dif is the immune factor responsible for the
profiles of several immune genes in Ae. aegypti antifungal immune defense in adult Drosophila
mosquitoes after infection with Gram (-) bacteria (12, 13). In this report, we have cloned and
(E. cloacae), Gram(+) bacteria (M. luteus), and characterized a homologue to Drosophila Dl from
fungal spores (B. bassiana). The expression levels the yellow fever mosquito Ae. aegypti, which we
of all tested immune genes were highly elevated named AaREL1. Although An. gambiae REL1
after any type of infection. However, the specific (Gambif1) has previously been cloned, the
7
conclusive in vivo data on its role in adult An. expression (39). With immune challenge, the over-
gambiae had been lacking (28). Our study has expression of Dif or Dl under a heat-shock
convincingly demonstrated that mosquito REL1 promoter rescued the lack of Drosomycin
serves as a functional Dif analog in adult Ae. inducibility in larval fat body cells of TW119
aegypti females. mutant flies with a deficiency uncovering both Dif
The AaREL1 expression profile is different and Dl genes (40). In contrast, the over-expression
from Drosophila Dl. In Drosophila, Dl transcripts of AaREL1-A fully up-regulated the Drosomycin
were previously shown to be markedly enhanced expression without immune challenge, and no
in adult males upon bacterial challenge (20). In a further up-regulation was found after challenge.
later study by Gross et al. (30), Drosophila Dl-B These results suggest that AaREL1 might not
transcript was increased upon bacterial challenge interact with some components of the Drosophila
in larvae and adult males. AaREL1 transcripts, Toll pathway. In Drosophila loss-of-function
however, were elevated in larvae and adult mutant of cactus, a Drosophila I-κB inhibitor
females after bacterial challenge. Moreover, the specific to Toll pathway, the Drosomycin genes
tissue-specific expression in female mosquitoes, are constitutively transcribed (14, 41), and the
the enriched ovary expression in the naïve state, over-expression of Drosophila Dl could fully
and the elevated fat body expression after septic activate the Drosomycin expression without
D
injury suggest AaREL1 as an immune factor of immune challenge (40). ow
n
female mosquitoes, as well as a morphogen during Due to the absence of Drosomycin lo
a
egg development. homologue in mosquitoes, we surveyed other de
d
Though many complications have been mosquito immune genes possibly regulated by fro
m
reported concerning specificity of response and Toll pathway. In general, the Drosophila immune h
gfuenngea rle-sgpuelcaitfioicn blya teIm din odru Tctoiolln p aothfw aDyrso (s3o8m),y cthine gspeenceisf icd eapcetinvdaetniot n upproonf ileT oblly pfautnhgwaal yc hsahlloewng ea. ttp://ww
w
expression and antifungal activity against some Drosomycin expression was shown to be partially .jb
fungi including, B. bassiana, are still two affected by the Imd pathway after bacterial c.o
representative characteristics of the Drosophila infection but to be regulated predominantly by the brg/
y
Toll immune pathway. We over-expressed Toll pathway during fungal infections (17, 23). In g
u
mosquito AaREL1-A in both in vivo and in vitro addition, the microarray analysis shows that the es
t o
Drosophila systems and examined the expression Toll pathway controls most of the late genes n
N
of Drosomycin in their native chromosomal induced by fungal infection (17). To find the ov
e
environment. Our results demonstrated that mosquito immune genes regulated by Toll m
b
e
AaREL1-A could fully up-regulate Drosomycin pathway, Ae. aegypti females were challenged by r 1
8
expression prior to bacterial challenge. In addition, different types of microorganisms (E. cloacae, M. , 2
0
the ectopic expression of AaREL1-A increased the luteus, and B. bassiana spore). The results 18
resistance against B. bassiana in transgenic flies. showed that the expression of Aedes Serpin-27A
In Drosophila S2 cells, the Drosomycin expression was elicited specifically by the fungal challenge,
was more affected by Dif, than by Dl (39). suggesting regulation by the Toll pathway similar
Moreover, Dif, not Dl, was sufficient to mediate to Drosophila Serpin-27A (37).
the induction of Drosomycin and antifungal The increased fungal susceptibility of
immunity against B. bassiana in mutant adult flies AaREL1 dsRNA-treated mosquitoes more clearly
(22, 23). The characteristics of AaREL1 in both a indicated that AaREL1 is an essential factor of the
Drosophila cell line and transgenic flies clearly Toll antifungal immune response. Our dsRNA
showed that AaREL1-A, a mosquito Dl knock-down results have demonstrated that
homologue, could function as Drosophila Dif in mosquito antifungal immune response by AaREL1
anti-fungal immune response. However, there can be distinguished from the immune response
were differences between AaREL1 and against Gram-negative bacteria mediated by
Drosophila Dif and/or Dl. The expression of Dif AaREL2, previously reported as a key regulator of
or Dl prior to LPS incubation caused a modest up- mosquito Imd pathway (42). Transgenic
regulation of Drosomycin expression in S2 cells, mosquitoes with stable, dominant, negative
and, after LPS treatment, further up-regulated the immune-deficient phenotype for AaREL2 showed
8
a marked susceptibility to Gram-negative bacteria and mosquito Ae. aegypti Aag2 cells, and the
infection, which severely compromised induction ectopic over-expression in transgenic flies provide
in the expression levels of both Defensin A and conclusive evidence to implicate AaREL1 as a Dif
Cecropin A. This indicated that the IMD pathway analog of the Toll-mediated antifungal immune
is generally conserved between Drosophila and pathway in adult female mosquitoes. In addition,
mosquitoes (42). Our RNAi experiments in vivo studies by the RNAi knockouts in
demonstrated the AaREL1 regulated antifungal mosquitoes strongly suggest that AaREL1 serves
immune response, distinctly separated from Imd as a key regulator of mosquito Toll immune
pathway immune response regulated by REL2. pathway but not as a redundant factor of the
In vitro comparative binding studies, immune response.
transfection assays in both Drosophila mbn-2 cells

REFERENCE

1. Beier, J. C. (1998) Annu. Rev. Entomol. 43, 519–543
D
2. Bremen, J. (2001) Am. J. Trop. Med. Hyg. 64, (Suppl.) 1–11. o
w
n
3. Beaty, B. (2000) Proc. Natl. Acad. Sci. USA, 97, 10295–10297. lo
a
d
e
d
4. Enserink, M. (2002) Science. 297, 1988–1989. fro
m
5. Wattam, A. R., and Christensen, B. M. (1992) Proc. Natl. Acad. Sci. USA, 89, 6502–6505. h
ttp
6. Curtis, C. F., and Lines, J. D. (2000) Parasitol. Today 16, 119–121. ://w
w
w
7. Lycett, G. J., and Kafatos, F. C. (2002) Nature 417, 387-388. .jb
c
.o
8. Holt, R. A. et al. (2002) Science 298, 129–149. rg
b/
y
9. Christophides, G. K. et al. (2002) Science 298, 159–165. g
u
e
s
10. Hultmark, D. (2003) Curr. Opin. Immunol. 15, 12–19. t o
n
N
o
11. Hoffmann, J. A. (2003) Nature 426, 33–38. v
e
m
b
12. Lemaitre, B., Reichhart, J. M., and Hoffmann, J. A. (1997) Proc. Natl. Acad. Sci USA 94, 14614–14619. er 1
8
13. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffman, J. A. (1996) Cell 86, 973–983. , 2
0
1
8
14. Levashina, E. A., Ohresser, D., Lemaitre, B., and Imler, J. L. (1998) J. Mol. Biol. 278, 515–527.
15. Hedengren, M., Asling, B., Dushay, M.S., Ando, I., Ekengren, S., Wihlborg, M., and Hultmark, D. (1999) Mol.
Cell 4, 827–837.
16. Leulier, F., Rodriguez, A., Khush, R.S., Chen, P., Abrams, J.M., and Lemaitre, B. (2000) EMBO Rep. 1, 353-
358.
17. De Gregorio, E., Spellman, P. T., Tzou, P., Rubin, G. M., and Lemaitre, B. (2002) EMBO J. 21, 2568–2579.
18. Drier, E. A., and Steward, R. (1997) Semin. Cancer Biol. 8, 83–92.
19. Pan, D., and Courey, A. J. (1992) EMBO J. 11, 1837–1842.
20. Reichhart, J. M., Georgel, P., Meister, M., Lemaitre, B., Kappler, C., and Hoffmann, J. A. (1993) C. R. Acad.
Sci. III 316, 1218–1224.
9
21. Ip, Y. T., Reach, M., Engstrom, Y., Kadalayil, L., Cai, H., Gonzales-Crespo, S., Tatei, K., and Levine, M.
(1993) Cell 75, 753–763.
22. Meng, X., Khanuja, B. S., and Ip, Y. T. (1999) Genes Dev. 13, 792–797.
23. Rutschmann, S., Jung, A. C., Hetru, C., Reichhart, J. M., Hoffmann, J. A., and Ferrandon, D. (2000) Immunity
12, 569–580.
24. Brennan, C. A., and Anderson, K. V. (2004) Annu. Rev. Immunol. 22, 457–483.
25. Qiu, P., Pan, P. C., and Govind, S. (1998) Development 125, 1909-1920.
26. Canton, Y., and Nappi, A. J. (2001) Immunogenetics 52, 157-164.
27. Osta, M. A., Christophides, G. K., Vlachou, D., and Kafatos, F. C. (2004) J. Exp. Biol. 207, 2551-2563.
28. Brillas-Mury, C., Charlesworth, A., Gross, I., Richamn, A., Hoffmann, J. A., and Kafatos, F. C. (1996) EMBO
J. 15, 4961–4701
29. Shin, S. W., Kokoza, V., Ahmed, A., and Raikhel, A. S. (2002) Proc. Natl. Acad. Sci. USA, 99, 9978–9983. D
o
w
n
30. Gross, I., Georgel, P., Oertel-Buchheit, P., Schnarr, M., and Reichhart, J. M. (1999) Gene 228, 233–242. lo
a
d
e
31. Hodel, M. R., Corbett, A. H., and Hodel, A. E. (2001) J. Biol. Chem. 276, 1317–1325. d fro
m
32. Markstein, M., Markstein, P., Markstein, V., and Levine, M. S. (2002) Proc. Natl. Acad. Sci. USA 99, 763–768. h
ttp
33. Samakovlis, C., Asling, B., Boman, H. G., Gateff, E., and Hultmark, D. (1992) Biochem. Biophys. Res. ://w
w
w
Commun. 188, 1169–1175. .jb
c
.o
34. Kappler, C., Meister, M., Lagueux, M., Gateff, E., Hoffmann, J. A., and Reichhart, J. M. (1993) EMBO J. 12, rg
b/
y
1561–1568. g
u
e
s
35. Lindmark, H., Johansson, K. C., Stoven, S., Hultmark, D., Engstrom, Y., and Soderhall, K. (2001) J. Immunol. t o
n
N
167, 6920–6923. o
v
e
m
36. Fallon, A. M., and Sun, D. (2001) Insect. Biochem. Mol. Biol. 31, 263–278. be
r 1
8
37. De Gregorio, E., Han, S. J., Lee, W. J., Baek, M. J., Osaki, T., Kawabata, S., Lee, B. L., Iwanaga, S., Lemaitre, , 2
0
1
8
B., and Brey, P. T. (2002) Dev. Cell 3, 581–592.
38. Brennan, C. A., and Anderson, K. V. (2004) Annu. Rev. Immunol. 22, 457-483.
39. Han, Z. S., and Ip, Y. T. (1999) J. Biol. Chem, 274, 21355–21361.
40. Manfruelli, P., Reichhart, J. M., Steward, R., Hoffmann, J. A., and Lemaitre, B. (1999) EMBO J. 18, 3380-
3391.
41. Nicolas, E., Reichhart, J. M., Hoffmann, J. A., and Lemaitre, B. (1998) J. Biol. Chem. 273, 10463–10469.
42. Shin, S. W., Kokoza, V., Lobkov, I., and Raikhel, A. S. (2003) Proc. Natl. Acad. Sci. USA 100, 2616–2621.
FIG. 1. Molecular characterization of two Aedes REL1 isoforms, AaREL1-A and AaREL1-B. A. Amino
acid sequence comparison of AaREL1-A and AaREL1-B to Drosophila Dl, Dl-B, and Anopheles REL1
(AgREL1). Alignments were done by ClustalW and were manually adjusted. C-terminal domains (CTDs) with a
Q-rich or Q/H-rich region that was shown to be important for transactivation in Drosophila Dl and Dif are
10