Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity

Abstract

Thor has been identified as a new type of gene involved in Drosophila host immune defense. Thor is a member of the 4E-binding protein (4E-BP) family, which in mammals has been defined as critical regulators in a pathway that controls initiation of translation through binding eukaryotic initiation factor 4E (eIF4E). Without an infection, Thor is expressed during all developmental stages and transcripts localize to a wide variety of tissues, including the reproductive system. In response to bacterial infection and, to a lesser extent, by wounding, Thor is up-regulated. The Thor promoter has the canonical NFkappaB and associated GATA recognition sequences that have been shown to be essential for immune induction, as well as other sequences commonly found for Drosophila immune response genes, including interferon-related regulatory sequences. In survival tests, Thor mutants show symptoms of being immune compromised, indicating that Thor may be critical in host defense. In contrast to Thor, Drosophila eIF4E is not induced by bacterial infection. These findings for Thor provide the first evidence that a 4E-BP family member has a role in immune induction in any organism. Further, no gene in the translation initiation pathway that includes 4E-BP has been previously found to be immune induced. Our results suggest either a role for translational regulation in humoral immunity or a new, nontranslational function for 4E-BP type genes.

Figure 1

Characterization of Thor sequences. (A) Restriction map of the genomic DNA surrounding the Thor P-element insertion (P-lacW) at polytene chromosome position 23F–24A. Arrangement of transcribed region and placement of insertion between the TATA box sequence and start codon also is indicated. Restriction enzyme abbreviations: G (BglII), P (PstI), H (HindIII), B (BamHI), and E (EcoRI). (B) Nucleotide sequence (top row) and predicted amino acid sequence (bottom row) of THOR. The start and stop codons are shown in bold with the intron represented by a dashed line. The predicted PolyA signal sequence is underlined. GenBank accession no. AF244353. (C) Amino acid alignment of THOR to 4E-BP sequences. Additional GenBank accession nos. are for human 4E-BP: h4E-BP1 (NM004095), h4E-BP2 (NM004096), and h4E-BP3 (NM003732); for mouse PHAS-I (U28656.1) and PHAS-II (U75530); for zebrafish: zf4E-BP3 (AI722723); Dictyostelium discoideum (C94507); and Schistosoma mansoni (AI014205).

Figure 2

Northern analysis of Thor expression. (A) Oregon R adults untreated (−), wounded (w) and infected (+). The control probe to show wounding and infection is Cecropin (Cec). The Thor probe is a DNA probe made from the 1.7-kb BglII fragment downstream of the P{lacW} insertion, as seen in Fig. 1A. Antisense RNA probes give the same results as DNA probes, and RNA probes were used for the results shown in B and C. The loading control probe is for actin, and the * refers to a transcript initially noticed because it changed the intensity of one of the actin bands. Reprobing confirmed hybridization of Thor to this 2-kb transcript (B and C). (B) Oregon R and mutant Thor¹ adults untreated (−) and with an infection (+). (C) Developmental profile of Thor expression in Oregon R. Stages are: 0–6 h (6), 6–12 h (12), and 12–24 h (24) embryos; first (1), second (2), and third (3) instar larvae; pupae (p); and adults (a). The blot was reprobed with rp49 as the loading control.

Figure 3

Tissue-specific localization of Thor expression. Thor expression is detected only in the central nervous system in embryos (A), but in many tissues in larvae and adults, e.g., the fat body (B), testes (B, *), and ring gland (C) of third instar larvae and the ovarian nurse cells (D) of adult females. Probes of A-C are digoxigenin-labeled, whereas D shows nuclear B-galactosidase staining of the Thor-enhancer trap as this shows more clearly that nurse cells as early as stage 4 express Thor.

Figure 4

Test for immune induction of eIF4E. A Northern blot of RNA from Oregon R adults untreated (−) and with an infection (+) was successively washed and reprobed for eIF4E, Thor and, as loading control, actin.

Figure 5

Thor upstream region. Regulatory motifs are outlined in the sequence 5′ of the TATA box (bold). Consensus sequences used to identify these motifs are as follows, and referenced in 14, except as indicated: NFκB response elements, GGGRNTYYYY (31); GATA, WGATAR; IL-6 response elements (NF-IL6), TKNNGNAAK, (IL-6-RE) CTGGGA; Nuclear factor endothelial leukocyte adhesion molecule 1 (NF-Elam1), WCAKCAK; Hepatic nuclear factor 5 response element (HNF5), TRTTTGY. A single mismatch in one of the NFκB elements is indicated by a lowercase letter. Additional sequences with core identity and at least a 0.8 outside core match to the transfac database (20) also are indicated. These Transfac sequences are the elements for: interferon regulatory factors IRF1, SNAAAGYGAAACC, and IRF2, GNAAAGYGAAASY; interferon-stimulated response element IRSE, CAGTTTCWCTTTYCC; signal transducers and activators of transcription, Drosophila STAT (D-STAT), TTTCCSGGAAA and vertebrate STAT (V-STAT), TTCCCRKAA; hepatic nuclear factor HNF1, GGTTAATNWTTAMMN; hepatocyte nuclear factor/forkhead homolog HNF3B, KGNANTRTTTRYTTW, HFH1, NAWTGTTTATWT and HFH8, TGTTTATNYR. The core sequences are underlined. Only the GATA near NFκB is indicated.

Figure 6

Survival of adults after bacterial infection. The survival rates (%) after injection of different types of bacteria for Thor flies (Thor¹ and Thor²) compared with imd flies and a wild-type revertant, Thor^1rv1 (wt). (A) Flies without an injection (−) and with a sterile wound (w) are presented for each stock as controls to compare the effect of infection. Bacteria used are E. cloacae B₁₂ (B) and E. coli (Ec). Additional types of bacteria used are presented separately in (B), S. epidermidis (Se), and (C), M. roseus (Mr). Flies were kept at 29°C, and data points are means and corresponding standard error for at least five replicates of samples of 20 flies.