The inducible expression of antimicrobial peptide genes in Drosophila melanogaster is regulated by the conserved Toll and peptidoglycan recognition protein LC/immune deficiency (PGRP-LC/IMD) signaling pathways. It has been proposed that the two pathways have independent functions and mediate the specificity of innate immune responses towards different microorganisms. Scattered evidence also suggests that some antimicrobial target genes can be activated by both Toll and IMD, albeit to different extents. This dual activation can be mediated by independent stimulation or by cross-regulation of the two pathways. We show in this report that the Toll and IMD pathways can interact synergistically, demonstrating that cross-regulation occurs. The presence of Spätzle (the Toll ligand) and gram-negative peptidoglycan (the PGRP-LC ligand) together caused synergistic activation of representative target genes of the two pathways, including Drosomycin, Diptericin, and AttacinA. Constitutive activation of Toll and PGRP-LC/IMD could mimic the synergistic stimulation. RNA interference assays and promoter analyses demonstrate that cooperation of different NF-B-related transcription factors mediates the synergy. These results illustrate how specific ligand binding by separate upstream pattern recognition receptors can be translated into a broad-spectrum host response, a hallmark of innate immunity.

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The Toll family of receptors is required for innate immune response to pathogen-associated molecules, but the mechanism of signaling is not entirely clear. In Drosophila the prototypic Toll regulates both embryonic development and adult immune response. We demonstrate here that the host protein Spä tzle can function as a ligand for Toll because Spä tzle forms a complex with Toll in transgenic fly extracts and stimulates the expression of a Tolldependent immunity gene, drosomycin, in adult flies. We also show that constitutively active mutants of Toll form multimers that contain intermolecular disulfide linkages. These disulfide linkages are critical for the activity of one of these mutant receptors, indicating that multimerization is essential for the constitutive activity. Furthermore, systematic mutational analysis revealed that a conserved cysteine-containing motif, different from the cysteines used for the intermolecular disulfide linkages, serves as a selfinhibitory module of Toll. Deleting or mutating this cysteinecontaining motif leads to constitutive activity. This motif is located just outside the transmembrane domain and may provide a structural hindrance for multimerization and activation of Toll. Together, our results suggest that multimerization may be a regulated, essential step for Toll-receptor activation.U pon infection, insects mount a rapid but rather nonspecific response. This insect antimicrobial response is similar to the mammalian innate immune response (1-7). The recognition receptors of the innate immune system have limited diversity and are designed to recognize pathogen-associated molecules such as lipopolysaccharides and peptidoglycans, which are common molecules found in groups of microorganisms. Toll and Toll-like receptors (TLRs) are a conserved family of recognition receptors in insects and mammals (5, 6). The Toll family of receptors is essential for innate immunity, but it is unclear how the receptors interact with microbial substances and host ligands.In Drosophila, the prototypic Toll receptor was first identified as an essential component for embryonic development (8). Subsequently, Toll was also shown to be essential for the induced expression of a subset of antimicrobial peptides during antifungal and anti-Gram-positive bacterial responses (9, 10). The regulation of the anti-Gram-negative bacterial response depends on a second pathway called the Immune deficiency (Imd) pathway, which controls the expression of another subset of antimicrobial peptides (2-4, 11). The induced expression of antimicrobial peptides through these two pathways is critical for Drosophila to survive microbial infections (12).The mechanism by which Toll receptors transmit the signal of infection across the cell membrane is not clear. Many components of the Toll pathway in the Drosophila innate immune response have been identified. The extracellular components include a peptidoglycan recognition protein (PGRP-SA), a serine protease (Persephone), a serine protease inhibitor (Necrotic), and a putative li...

The innate immune response in Drosophila involves the inducible expression of antimicrobial peptide genes mediated by the Toll and IMD signaling pathways. Dorsal and DIF act downstream of Toll, whereas Relish acts downstream of IMD to regulate target gene expression. Dorsal, DIF, and Relish are NF-κB-related transcription factors and function as obligate dimers, but it is not clear how the various dimer combinations contribute to the innate immune response. We systematically examined the dimerization tendency of these proteins through the use of transgenic assays. The results show that all combinations of homo-and heterodimers are formed, but with varying degrees of efficiency. The formation of the DIFRelish heterodimer is particularly interesting because it may mediate signaling for the seemingly independent Toll and IMD pathways. By incorporating a flexible peptide linker, we specifically tested the functions of the DIF^Relish (a^sign represents the peptide linker) linked heterodimer. Our results demonstrate that the linked heterodimer can activate target genes of both the Toll and IMD pathways. The DIF and Relish complex is detectable in whole animal extracts, suggesting that this heterodimer may function in vivo to increase the spectrum and level of antimicrobial peptide production in response to different infections.IMD | innate immunity | Toll