NF-κB/Rel proteins and the humoral immune responses of Drosophila melanogaster

Abstract

Nuclear Factor-κB (NF-κB)/Rel transcription factors form an integral part of innate immune defenses and are conserved throughout the animal kingdom. Studying the function, mechanism of activation and regulation of these factors is crucial for understanding host responses to microbial infections. The fruit fly Drosophila melanogaster has proved to be a valuable model system to study these evolutionarily conserved NF-κB mediated immune responses. Drosophila combats pathogens through humoral and cellular immune responses. These humoral responses are well characterized and are marked by the robust production of a battery of anti-microbial peptides. Two NF-κB signaling pathways, the Toll and the IMD pathways, are responsible for the induction of these antimicrobial peptides. Signal transduction in these pathways is strikingly similar to that in mammalian TLR pathways. In this chapter, we discuss in detail the molecular mechanisms of microbial recognition, signal transduction and NF-κB regulation, in both the Toll and the IMD pathways. Similarities and differences relative to their mammalian counterparts are discussed, and recent advances in our understanding of the intricate regulatory networks in these NF-κB signaling pathways are also highlighted.

Fig. 1

Drosophila Toll pathway. Drosophila Toll pathway is a cytokine receptor pathway that responds indirectly to microbial infection. Distinct circulating receptor proteins recognize different microbial structures or activities and activate a protease cascade, which culminates in the processing of Spätzle. Lys-type PGN, from bacterial cell walls, is recognized by PGRP-SA/PGRP-SD and GNBP1, while GNBP3 detects β-(1, 3)-glucan from fungal cell walls. These upstream recognition events trigger a proteolytic cascade which proceeds through the serine proteases ModSP and Grass to activate Spätzle Processing Enzyme (SPE) which in turn cleaves pro-Spätzle to release the mature Toll ligand, Spätzle. In addition, virulence-associated proteases, released by pathogens, cleave and activate another pathway that requires the protease Persephone and converges on the activation of SPE. The intracellular Toll signaling pathway is very homologous to the mammalian MyD88-dependent signaling pathway. It signals through an upstream complex containing the adaptor proteins dMyD88, Tube and the kinase Pelle (an IRAK homolog). Pelle triggers the phosphorylation, K48-ubiquitination and proteasomal degradation of the IκB protein, Cactus, thereby releasing the Drosophila NF-κB/Rel factors DIF/Dorsal to translocate into the nucleus and transcribe target AMP genes, such as Drosomycin

Fig. 2

Drosophila IMD pathway. Recognition of DAP-type PGN by the cell surface receptor PGRP-LC or the cytosolic receptor PGRP-LE triggers the IMD pathway. DAP-type PGN forms the cell wall of most Gram-negative bacteria and some Gram-positive bacteria, such as Bacillus spp. and Listeria. Extracellularly, detection of polymeric and monomeric PGN (TCT) occurs through homo or heterodimers of PGRP-LC splice forms, as depicted. PGRP-LE detects intracellular bacteria and stimulates the IMD pathway as well as an autophagic response. PGRP-LC and PGRP-LE signaling proceeds through IMD, FADD and the caspase DREDD, which are likely to form a complex on the activated receptor. DREDD-dependent cleavage of IMD exposes an IAP-binding motif in IMD, through which it binds DIAP2. This IMD-DIAP2 association leads to robust K63-ubiquitination of cleaved IMD by DIAP2, Uev1a, Bendless (Ubc13) and Effete (Ubc5). These K63-polyubiquitin chains are likely to serve as a scaffold for the recruitment and activation of the TAK1 kinase complex and the IKK complex. Once activated, TAK1 in turn activates both IKK and JNK kinase. Relish is phosphorylated by IKK and cleaved by DREDD to release the fully active N-terminal RHD for translocation into the nucleus and the transcriptional induction of AMP genes and other targets. The concurrent activation of JNKK leads to the phosphorylation and nuclear translocation of AP-1

Fig. 3

κB site specificity of Drosophila NF-κB proteins: NF-κB factors, upon activation by the Toll and IMD pathways, translocate into the nucleus and bind to specific κB sites in the promoter/enhancer region of immune responsive AMP genes. DIF and Dorsal are activated by Toll signaling through degradation of the IκB inhibitor protein Cactus, while Relish activation is triggered by IMD signaling through DREDD-dependent endoproteolytic cleavage to remove the C-terminal IκB-like domain and the IKK-mediated phosphorylation of serines 528 and 529. In this figure, selected AMP genes Drosomycin,Diptericin, AttacinA and Metchnikowin with their proximal DIF, Dorsal or Relish binding κB sites and Serpent (Srp) binding GATA sites are represented (Senger et al. 2004; Busse et al. 2007; Tanji et al. 2007). Expression of AttacinA requires both DIF and Relish, and hence contains separate binding sites for each of them, while Diptericin requires only Relish and has two Relish specific sites. Drosomycin and Metchnikowin each contain a distinct DIF/Relish site, which is responsive to both DIF and Relish. It is not clear if this site binds a heterodimer (as suggested by this figure) or if it functions by binding either homodimer. In addition, the Metchnikowin enhancer includes separate Relish-specific sites, while Drosomycin has another DIF/Dorsal-specific site only. Refer to Sect. 4.3 in the text for details on the consensus κB and GATA sites and the complex regulation involved in the expression of AMPs