DEAF-1 regulates immunity gene expression in Drosophila

Abstract

Immunity genes are activated in the Drosophila fat body by Rel and GATA transcription factors. Here, we present evidence that an additional regulatory factor, deformed epidermal autoregulatory factor-1 (DEAF-1), also contributes to the immune response and is specifically important for the induction of two genes encoding antimicrobial peptides, Metchnikowin (Mtk) and Drosomycin (Drs). The systematic mutagenesis of a minimal Mtk 5′ enhancer identified a sequence motif essential for both a response to LPS preparations in S2 cells and activation in the larval fat body in response to bacterial infection. Using affinity chromatography coupled to multidimensional protein identification technology (MudPIT), we identified DEAF-1 as a candidate regulator. DEAF-1 activates the expression of Mtk and Drs promoter-luciferase fusion genes in S2 cells. SELEX assays and footprinting data indicate that DEAF-1 binds to and activates Mtk and Drs regulatory DNAs via a TTCGGBT motif. The insertion of this motif into the Diptericin (Dpt) regulatory region confers DEAF-1 responsiveness to this normally DEAF-1-independent enhancer. The coexpression of DEAF-1 with Dorsal, Dif, and Relish results in the synergistic activation of transcription. We propose that DEAF-1 is a regulator of Drosophila immunity.

Transcriptional regulation of Drosophila antimicrobial genes depends on Rel and GATA transcription factors (1–6). Many immunity genes contain tightly linked Rel- and GATA-binding sites in promoter-proximal regions. GATA sites are important for establishing responses in distinct tissues such as the fat body and midgut. Serpent (dGATAb) is thought to regulate antimicrobial gene expression in the fat body (7, 8), whereas dGATAe activates such genes in the midgut in response to ingested microbes (9). In contrast, Dorsal, Dif, and Relish, the NF-κB homologues in flies, shuttle between the cytoplasmic and nuclear compartments, acting as “on/off switches” for induction (10–12). Additional factors, such as HOX and POU domain proteins, bind to distal enhancer elements and maintain constitutive domains of gene activity (13). A regulatory element (R1) also has been described within the CecA1 enhancer (14), although the factor that interacts with this motif is unknown.

Deformed epidermal autoregulatory factor-1 (DEAF-1) is a transcription factor that was originally shown to bind the autoregulatory enhancer of the Deformed (Dfd) Hox gene, which is activated in embryonic head segments of Drosophila (15). DEAF-1 recognizes several TTCG motifs within the portion of the Dfd autoregulatory region termed “module E.” In addition, DEAF-1 binds several similar motifs within a Dfd response element (DRE) from the 1.28 gene that enhances maxillary gene expression during embryogenesis (16). The DEAF-1 binding elements identified in these studies are reportedly not required for enhancer activity however (16, 17).

The 576-aa DEAF-1 protein possesses two conserved domains, SAND and MYND. The 113-aa SAND domain (named for SP100, AIRE-1, NucP41/75, and DEAF-1) (18) is responsible for DNA binding via a highly conserved KDWK peptide motif (19, 20). The 32-aa MYND domain (for myeloid, Nervy, and Deaf-1) contains non-DNA-binding zinc fingers that are thought to mediate protein–protein interactions (15). DEAF-1 is maternally expressed, and the encoded protein is broadly distributed throughout the early embryo. It exhibits augmented expression in the CNS after stage 14 (15). Zygotic mutants develop to pupal stages, but do not eclose, whereas maternal mutants display severe defects in early embryonic patterning (21). Overexpression of DEAF-1 by using a maternal driver inhibits germ-band retraction and causes defects in dorsal closure, whereas overexpression at later stages causes cell death (21).

In vertebrates, the closest relatives of DEAF-1 are nuclear DEAF-1-related factor (NUDR) and Suppressin (SPN) (22, 23). Both factors are expressed in a wide variety of tissue types. NUDR functions to either activate (22) or repress (24) transcription depending on its context, and it binds sequences bearing TTCGGG or TTTCCG motifs (22). SPN does not have a characterized role in transcription. It was originally identified as a protein secreted by the bovine pituitary gland that, when added to tissue culture media, inhibits splenocyte proliferation (25) and stimulates IFN-α/β production in leukocytes (26).

Previous studies identified a 208-bp proximal enhancer that regulates the expression of the Mtk gene. This enhancer directs high levels of transcription in the fat bodies of infected larvae and also is induced by LPS preparations in S2 cells. These regulatory activities depend on a cluster of Rel- and GATA-binding sites. Here, we present evidence that an additional sequence motif (E8) contributes to Mtk activation. Enhancer DNA affinity chromatography assays and proteomic analysis identified DEAF-1 as a protein that interacts with the E8 motif. DEAF-1 binds to the consensus sequence TTCGGBT, which is contained within the E8 region of the Mtk enhancer. Additional DEAF-1 consensus motifs are found in the regulatory regions of other immunity genes, such as Drosomycin (Drs). Evidence is presented that DEAF-1 works synergistically with Dorsal, Dif, and Relish to induce gene expression in response to LPS. We propose that DEAF-1 is an essential component of the immune response in Drosophila.

Results and Discussion

Identification of a cis-Regulatory Element in the Mtk Enhancer.

To identify regulatory motifs within the minimal 208-bp Mtk enhancer, we scrambled the nucleotide sequences of 11 regions (E1–E11) flanking the previously identified Rel- and GATA-binding sites (Fig. 1A). Several of these scrambled elements (SEs) were found to alter the activities of Mtk-Luciferase (Mtk-Luc) promoter-reporter fusion genes in transient transfection assays with S2 cells in the presence of LPS (Fig. 1B). It should be emphasized that this assay does not measure a response to LPS, but rather to Gram-negative peptidoglycans that commonly occur in commercial preparations of LPS (27, 28). Gram-negative peptidoglycans have been shown to signal through the Imd and, to a lesser degree, the Toll pathway (27), so this assay probably reflects both types of signaling cascades. Fusion genes bearing scrambled sequences in region 4, 5, or 6 are roughly twice as active as the native enhancer, suggesting a disruption of potential repressor elements. A 50% reduction in induction is seen for the fusion gene containing scrambled sequences in region 9, suggesting the loss of a weak activator element. Most notably, there is a severe 21-fold decrease in the induced expression of the Mtk-Luc fusion gene containing scrambled sequences in region 8 (SE8). There also is a 3-fold reduction in the constitutive activities of this fusion gene. Thus, region 8 appears to contain an essential activator element.

Fig. 1.

Mutation of the Mtk regulatory sequence reveals functional motifs. (A) Diagram of the 208-bp Mtk regulatory sequence. Known Rel and GATA factor-binding sites are boxed in gray. The transcription start site determined by 5′ RACE (FirstChoice RLM-RACE kit; Ambion) is to the right of the arrow. Elements selected for mutation (E1–E11) are underlined. (B) The region in A bearing no alterations (WT) or bearing scrambled elements (SE1–SE11) was cloned into a luciferase reporter construct that was then transiently transfected into S2 cells. After a 6-h induction with LPS (10 μg/ml O127:B8; Sigma), cells were harvested and luciferase activity was measured. Error bars correspond to one standard deviation of the mean (SDM). (C) Transgenic larvae carrying Mtk-lacZ (Upper) or Mtk SE8-lacZ (Lower) constructs were generated via P-element insertion. Third instar transgenic larvae were infected with a mixture of E. coli and M. luteus and stained for lacZ 6 h later. Shown is a closeup of the fat body. (D) Transgenic Mtk-lacZ (Upper) and Mtk SE8-lacZ (Lower) larvae were fed E. carotovora and dissected 6 h later. The intact transgene produces constitutive activity in the proventriculus and inducible activity in the anterior midgut. The transgene carrying the SE8 mutation shows no detectable activity in either tissue (Lower).

To test the activities of the E8 sequence in vivo, we examined transgenic larvae carrying an Mtk SE8-LacZ transgene. Upon septic injury with a mixture of Escherichia coli and Micrococcus luteus, the wild-type Mtk-lacZ transgene drives intense lacZ expression in the fat body (Fig. 1C Upper). Mutation of E8 essentially abolishes reporter gene expression in three of four independent lines and allows only a weak response in the fourth (Fig. 1C Lower) (data not shown). The wild-type Mtk-lacZ fusion gene is constitutively active in the posterior proventriculus of most larvae and in the anterior midgut of ≈20% of the larvae. Upon ingestion of Erwinia carotovora, there is at least a doubling in the number of larvae that exhibit expression in the anterior midgut (Fig. 1D Upper). In contrast, the Mtk SE8-LacZ transgene completely lacks both constitutive and induced activity throughout the midgut in three of four transgenic lines, with a weak response in the fourth line (Fig. 1D Lower) (data not shown).

Characterization of DEAF-1-Recognition Sequences.

We used a proteomics approach to identify proteins that bind E8. The entire Mtk regulatory domain (WT and SE8) was biotinylated and coupled to magnetic Dynal beads (Fig. 2A). An EcoRI restriction site was included proximal to the biotin moiety. Dynal bead–DNA complexes were incubated with nuclear extracts from S2 cells that had been treated with LPS. The resulting nucleoprotein complexes were washed extensively and eluted with a brief EcoRI digest. Eluted proteins were then subjected to Multidimensional Protein Identification Technology (MudPIT) analysis (29).

Fig. 2.

Element 8 binds DEAF-1. (A) Schematic of the pulldown assay. Wild-type and SE8 regulatory sequences were PCR-amplified by using a 5′ biotinylated primer and coupled to magnetic Dynal beads. An EcoRI site was included between the biotin moiety and Mtk sequences. The bead–DNA complexes were blocked with BSA and incubated with nuclear extracts from S2 cells induced with LPS. After extensive washing, the nucleoprotein complexes were eluted with an EcoRI digest and subjected to MudPIT analysis. (B) Recombinant His-tagged DEAF-1 protein was incubated with radiolabeled E8 (CATTCATTCGGCTGC) and SE8 (CATACGTGTCCTTGC) oligos, and complexes were resolved on 6% native acrylamide gels. Protein amounts range from 10–300 ng. (C) Briefly, 200 ng of DEAF-1 was incubated with 6 pmol of radiolabeled E8 oligo (lanes 1–5) and then challenged with a 3× or 10× excess of cold SE8 (lanes 2 and 3) or E8 (lanes 4 and 5) oligos. (D) Position weighted matrix for DEAF-1 derived from published footprinting data (16) and SELEX assays (see Fig. S1). (E) DEAF-1 protein was bound to radiolabeled E8 oligo and challenged with a 3×, 10×, or 30× excess of cold oligos bearing single changes in the DEAF-1 consensus indicated in D.

MudPIT identified several candidate proteins that were uniquely present in the eluate from the native Mtk regulatory sequence and not from the SE8 mutant enhancer sequence. One of these, DEAF-1, was particularly interesting because it recognizes a sequence motif, TTCG (15, 16), which resembles the E8 sequence (TCATTCGGC). This led us to pursue the role of DEAF-1 in regulating Mtk expression.

To test whether DEAF-1 recognizes Mtk regulatory sequences, gel shift assays were performed with increasing amounts of recombinant DEAF-1 protein and radiolabeled E8 and SE8 oligonucleotides (Fig. 2B). The DEAF-1 protein binds to E8 (lanes 1–4), but not the SE8 scrambled sequence (lanes 5–8). Competition assays were done by incubating DEAF-1 with the radiolabeled E8 sequence, followed by the addition of an excess of unlabeled E8 or SE8 oligonucleotides (Fig. 2C). A 10-fold excess of unlabeled E8 removes DEAF-1 from the radioactive probe (compare lane 5 with lane 1), whereas the same amount of the SE8 oligonucleotide only weakly disrupts binding (compare lane 3 with lane 1).

The previously published DEAF-1-binding site, TTCG, is based on footprint assays using the Dfd autoregulatory enhancer and the 1.28 gene enhancer (15, 16). This analysis was extended by performing systematic evolution of ligands by exponential (SELEX) enrichment experiments (30). Recombinant His-tagged DEAF-1 protein was incubated with a random library of radiolabeled oligonucleotides. Protein–DNA complexes were gel-purified and PCR-amplified, and the selected DNA was subjected to two additional rounds of binding and amplification. The DNAs were sequenced and aligned with the published footprint data (16) to generate a position-weighted matrix [Fig. 2D and supporting information (SI) Fig. S1]. The broadest consensus sequence using this approach is TTCGGBT. The SELEX data show a weaker preference for cytosine at position 3 than the previous footprinting data and a stronger selection for guanine at position 5. This finding may reflect differences in the binding of DEAF-1 to individual sites, compared with the clustered sites seen in the Dfd enhancer.

The strongest selection by DEAF-1 occurs at positions 2–5 (TCGG). We verified the importance of each position by performing gel shift competition assays (Fig. 2E). DEAF-1–E8 complexes were incubated with unlabeled oligonucleotides bearing mutations at each position along the consensus binding sequence. Oligonucleotides bearing mutations at position 1, 6, or 7 (TTCGGBT) successfully competed with radiolabeled E8 for DEAF-1 binding, suggesting that they contain an intact core-binding sequence (compare lanes 5–7 and 21–26 with lane 1). In contrast, mutations at positions 2–5 (TTCGGBT) greatly impaired competitive binding of the modified oligonucleotides (compare lanes 8–20 with lane 1). Hence, strong DEAF-1-binding sites appear to contain a TCGG core sequence.

DEAF-1 Activates Mtk Expression.

Transient transfection assays were done to investigate the ability of DEAF-1 to activate transcription in S2 cells. DEAF-1 was expressed in S2 cells by placing the DEAF-1 coding sequence under the control of the actin promoter (pMA6-DEAF-1). This DEAF-1 expression vector was cotransfected with an Mtk-Luc reporter construct in S2 cells (Fig. 3A). The fusion gene is normally induced 12-fold upon addition of LPS to the culture medium. The addition of pMA6-DEAF-1 causes a 5-fold increase in the basal expression of the Mtk-Luc reporter gene and a 28-fold increase upon addition of LPS. In contrast, an Mtk-Luc reporter construct bearing the scrambled E8 sequence (Mtk SE8-Luc) did not respond to expression of DEAF-1.

Fig. 3.

DEAF-1 stimulates transcription of immunity genes. (A) Diagram of the 208-bp Mtk regulatory sequence with approximate location of Rel- (red), GATA- (green), and DEAF-1 (blue)-binding sites is noted. The red diamond denotes a bidirectional Rel site. The striped triangle represents an additional consensus DEAF-1 site that overlaps the second GATA site. The effect of cotransfecting pMA6-DEAF-1 (2 μg) with either wild-type or SE8 templates is shown in the bar graph. (B) Diagram of a 746-bp Drs regulatory sequence containing close matches to the DEAF-1 consensus. The Drs transcription start site was mapped by using 5′ RACE (FirstChoice RLM-RACE kit; Ambion) to the sequence CCAAGCCACAAGTCG (starting nucleotide underlined). One DEAF-1 site (striped triangle) overlaps a Rel site. The five nonoverlapping DEAF-1 sites (E8.1–E8.5) were mutagenized and tested for a response to transfected pMA6-DEAF-1 (2 μg). (C) Gel shift showing DEAF-1 binding to normal and scrambled recognition elements from B. (D) The E8 motif from Mtk was inserted into the Dpt regulatory region in either a forward (FW) or reverse (RV) orientation relative to the promoter. For comparison, the SE8 sequence also was inserted (white triangle). Error bars correspond to one SDM.

We surveyed other immunity genes for sequences that conformed to the DEAF-1-binding consensus. The 746-bp 5′ enhancer of Drs (6) contains five potential DEAF-1-binding sites (E8.1–E8.5) (Fig. 3B). Each site binds DEAF-1 with a different affinity (Fig. 3C); sites E8.3 and E8.4 bind particularly well. The five recognition sequences were scrambled in the context of an otherwise normal Drs enhancer and analyzed in S2 cells by using a Drs-Luc reporter construct (Fig. 3B). A Drs-Luc reporter containing wild-type sequences is induced ≈2-fold upon LPS addition. Cotransfection with the pMA6-DEAF-1 expression vector causes an additional 12-fold increase in Drs-Luc reporter gene expression in the absence of LPS and a 17-fold increase with LPS. Mutations in individual binding sites reduce both basal and activated transcription. Mutation of site 8.3 causes the most dramatic reduction in activity.

The 201-bp Diptericin enhancer mediates strong expression in the fat bodies of infected larvae, but is only weakly induced in S2 cells (≈2-fold) (Fig. 3D). The enhancer likely lacks DEAF-1-binding sites based on DNA sequence analysis, and a Dpt-Luc reporter construct responds only weakly to transfected DEAF-1 in S2 cells. To determine whether insertion of a DEAF-1 consensus binding site can confer an ability to respond to DEAF-1 expression, we placed a single, optimal DEAF-1 site 10 bp upstream of the endogenous GATA motif, either in the forward or reverse orientation. Both constructs exhibit a significant increase in luciferase expression when coexpressed with a DEAF-1 expression construct, compared with the unmodified reporter fusion gene. In contrast, insertion of the scrambled E8 (SE8) sequence led to only a modest increase in reporter gene expression.

Deaf-1 Synergizes with Dorsal, Dif, and Relish.

Mutation of all three Rel sites or all three GATA sites within the Mtk enhancer causes a complete loss of induced activity in S2 cells (6). Thus, Rel and GATA factors function synergistically to activate immunity gene expression. The presence of DEAF-1-binding sites near the Rel and GATA sites suggests that it may cooperate with these factors during the mounting of an immune response. To test this we cotransfected pMA6-DEAF-1 with expression vectors for Dorsal, Dif, and Relish. Both Mtk-Luc and Drs-Luc fusion genes were used as reporters to monitor the combinatorial activities of these transcription factors (Fig. 4 A–F).

Fig. 4.

Synergy between DEAF-1 and Rel factors. (A–C) The Mtk-Luc fusion gene was transfected with pMA6-DEAF-1 in the absence or presence of increasing amounts of pMA6-Dorsal (A), pMA6-Dif (B), or pMA6-Relish (C) both with and without the addition of LPS as noted. The average of three experiments is shown. Fold synergy is shown in italics. (D–F) Same as A–C, except the Drs-Luc fusion gene is monitored for reporter expression. Error bars correspond to one SDM.

The Mtk-Luc reporter gene (Fig. 4 A–C) is induced 12-fold with LPS. Addition of 1 μg of pMA6-DEAF-1 raised the basal activity 5-fold and boosts the response to added LPS ≈30-fold. Separate transfections with individual Dorsal (Fig. 4A), Dif (Fig. 4B), and Relish (Fig. 4C) expression vectors also result in substantial activation (up to 67-fold for Dorsal, 166-fold for Dif, and 350-fold for Relish with LPS). Cotransfection of pMA6-DEAF-1 with each Rel factor results in some degree of synergy, denoted in italics above each bar graph. The level of synergy is calculated by dividing the activity of two factors working together by the sum of their individual activities. The average fold synergy with DEAF-1 on the Mtk-Luc reporter is 1.6-fold with Dorsal, 2.2-fold with Dif, and 1.7-fold with Relish.

Similar results were obtained by using the Drs-Luc reporter (Fig. 4 D–F), which is induced an average of 2.8-fold upon addition of LPS in S2 cells. The addition of 1 μg of pMA6-DEAF-1 raises basal activity 6-fold and LPS induction 10-fold. Separate transfections of either Dorsal or Dif dramatically activate this reporter (up to 60-fold with LPS), whereas Relish appears to be a much weaker activator (7-fold with LPS). Cotransfection of pMA6-DEAF-1 with each Rel factor results in an average of 3-fold synergy with Dif and 1.9-fold synergy with Dorsal. Only additive effects (average 1.2-fold synergy) were obtained with Relish. Altogether, these results suggest that DEAF-1 differentially augments the activities of different Rel factors during the induction of immunity genes. Particularly strong synergy is seen between DEAF-1 and Dif.

In summary, we have presented evidence that DEAF-1 is an essential component of the immune response in both Drosophila larvae and S2 cells. It appears to augment the synergistic activities of Rel and GATA transcription factors during the immune response. In so doing, it provides a signal amplification mechanism so that certain innate immunity genes, such as Mtk, can be transcribed at particularly high levels while using the same signaling pathways as other less highly expressed immunity genes. The critical E8 motif, TTCGGCT, is highly conserved among the Mtk enhancers of divergent Drosophilids and is closely linked to a paired set of Rel and GATA sites. It is therefore conceivable that DEAF-1 facilitates the binding or transcriptional efficacy of Rel and GATA factors at linked sites.

The requirement for DEAF-1 in the regulation of Drs, but not Dpt, hints at a possible function for DEAF-1 in Toll signaling. Cotransfection experiments using Mtk-Luc and Drs-Luc reporter genes demonstrate that DEAF-1 synergizes with Dorsal and especially Dif, two effectors of Toll signaling. Only weak cooperation occurs between DEAF-1 and Relish, a target of the Imd pathway. Microarray studies of flies mutant for Toll and Imd pathway components (31) have comprehensively identified groups of genes coregulated by each pathway. Interestingly, several genes that require Toll signaling for regulation, such as Cactus, IM1, and Dif, contain one perfect and several near-perfect consensus DEAF-1-binding sites within 1 kb of the transcription start sites. Future studies should determine whether DEAF-1 is a constitutive component of immune tissues like the GATA factors or is regulated in response to infection by Toll signaling as seen for the Rel factors.

Methods

Site-Directed Mutagenesis.

Primers carrying the desired mutation plus 15–20 nt flanking each end were phosphorylated in 10 mM Tris (pH 8.0), 1× T4 ligase buffer (New England BioLabs), 1 unit/μl polynucleotide kinase (New England BioLabs), and 5 pmol/μl primer for 1 h at 37°C. Then 10 pmol of phosphorylated primer was used in a PCR containing 50 ng template, 1× Pfu Turbo buffer (Stratagene), 0.2 mM dNTP mix, and 0.1 unit/μl Pfu Turbo polymerase (Stratagene). Thermocycling parameters were 1 min at 94°C, 30 cycles at 94°C for 1 min, 55°C for 1 min, 65°C for 10 min, ending at 15°C. PCR products were DpnI-digested (1.6 units/μl; NEB) for 1.5 h, transformed into DH5α cells, and screened for mutations by sequencing. The scrambled Mtk sequences indicated in Fig. 1A are as follows: Mtk SE1, GCAGCG; Mtk SE2, GGTTTG; Mtk SE3, GCGTTGG; Mtk SE4, ACGCTAGCA; Mtk SE5, CGCTCGCC; Mtk SE6, TCAGCTGA; Mtk SE7, TCGCATCC; Mtk SE8, ACGTGTCCT; Mtk SE9, AGACAATCAG; Mtk SE10, CGTCGACTA; and Mtk SE11, TGTCTGCTGT. The wild-type and scrambled Drs sequences are as follows: E8.1/SE8.1, ATCGGTG/CTAGGTG; E8.2/SE8.2, TTCGGTA/GACTGTT; E8.3/SE8.3, TACCGAA/CAGTAAC; E8.4/SE8.4, ACCCGAC/CACGACC; and E8.5/SE8.5, ATCGGCT/ACGTTCG.

Affinity Chromatography and MudPIT.

The wild-type or SE8 Mtk regulatory region from −208 to +33 was PCR-amplified with a biotinylated 5′ primer (ELIM Biopharmaceuticals) harboring an EcoRI site (5′-biotin-AAAAGAATTCTAGGCTGATAATCCGGGACCGTGGGAAGTC-3′) and unlabeled 3′ primer (5′-TGCATCTTAGCTCGGTGGCGGGAATTGATTG). Then 40 pmol PCR product was coupled to 2 mg of Dynabeads (Dynal Biotech) per the manufacturer's instructions. DNA–bead complexes were blocked for 1 h at 4°C in BC-100 buffer [20 mM Hepes (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA (pH 8.0), 0.5 mM PMSF, 0.5 mM DTT] plus 5% BSA, 0.1 mg/ml salmon sperm DNA, and 0.01% Triton X-100. Nuclear extracts (1 mg) from S2 cells induced 6 h with LPS (10 μg/ml O127: B8; Sigma) were added and incubated for 2 h at 4°C. Protein–DNA complexes were washed one time with blocking buffer and six times with BC-100 containing no PMSF, plus 0.1 mg/ml salmon sperm DNA and 0.01% Triton X-100. Complexes were eluted from the Dynal beads with a 10-min EcoRI digest (1 unit/μl FPLC pure; Amersham) at 37°C. Eluted protein complexes were precipitated by the addition of trichloroacetic acid and then digested by the sequential addition of lys-C and trypsin proteases as described previously (32). The digested samples were then separated by using an online multidimensional seven-step chromatographic strategy, followed by tandem mass spectrometric analysis of the fractionated peptides as they eluted directly into a LTQ-Orbitrap mass spectrometer (Thermofisher) (33). Data analysis was performed by using the SEQUEST and DTASelect2 algorithms, and peptide identifications were filtered by using a false-positive rate of <5% as estimated by using a decoy database strategy (34–36).

Fly Strains and Infections.

All flies were maintained at 25°C on standard cornmeal medium. The method for creating transgenic lines and causing septic infection was described in ref. 6. The method for E. carotovora infection was described in ref. 9. At least three independent lines were analyzed per experiment.

Cell Culture and Transfections.

S2 cells (Invitrogen) were maintained at 25°C in Schneider's Insect Media supplemented with 10% heat-inactivated FBS and 1× Pen/Strep (Sigma). For transfections, cells were seeded in 24-well plates at a density of 2 × 10⁶ cells per ml. One day after plating, transfections were carried out by using the calcium phosphate method (37). Two days after transfection, 10 μg of LPS (O127:B8; Sigma) was added, and the cells were harvested 6 h later. Cells were lysed (1× reporter lysis buffer; Promega), and luciferase activity was measured on a TD-20/20 luminometer (Turner Designs). The amount of reporter DNA was 200 ng in each experiment.

SELEX and EMSA assays.

SELEX and EMSA assays were performed as described previously (6).