Drosophila rely entirely on an innate immune response to combat microbial infection. Diaminopimelic acid-containing peptidoglycan, produced by Gram-negative bacteria, is recognized by two receptors, PGRP-LC and PGRP-LE, and activates a homolog of transcription factor NF-kappaB through the Imd signaling pathway. Here we show that full-length PGRP-LE acted as an intracellular receptor for monomeric peptidoglycan, whereas a version of PGRP-LE containing only the PGRP domain functioned extracellularly, like the mammalian CD14 molecule, to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface. Interaction with the imd signaling protein was not required for PGRP-LC signaling. Instead, PGRP-LC and PGRP-LE signaled through a receptor-interacting protein homotypic interaction motif-like motif. These data demonstrate that like mammals, drosophila use both extracellular and intracellular receptors, which have conserved signaling mechanisms, for innate immune recognition.

Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides, through two NF-κB signaling pathways - IMD and Toll. The IMD pathway is triggered by DAP-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan receptors is thought to involve K63-ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. We now show that PGN-stimulation causes caspase-mediated cleavage of the imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N-terminus. A functional IBM is required for the association of cleaved-IMD with the ubiquitin E3 ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63-ubiquitination in immune-induced NF-κB signaling.

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.

Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP-LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid-regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP-LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals.

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of Gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.Innate immune defenses against pathogens are initiated by pattern recognition receptors that bind conserved stereotypical, rather than particular, molecular structures present in a wide spectrum of microorganisms but absent in the host (1). A representative example of such structures is peptidoglycan (PGN), 3 the major constituent of the cell wall of both Gram-positive and -negative bacteria. The peptidoglycan recognition protein (PGRP) family is a class of pattern recognition receptors that bind, and sometimes cleave, PGN. A total of 13 and four PGRP family members have been identified in Drosophila and humans, respectively (2-5). PGRPs are often characterized based on their polypeptide length. Short form PGRPs, such as PGRP-SA and -SD, contain a single PGRP domain (ϳ180 amino acids) and, in most cases, a signal sequence, leading to the secretion of the proteins. Long form PGRPs, such as PGRP-LC and -LE, contain other domain(s) in addition to the PGRP domain, often including a transmembrane region (6). The PGRP domain is similar in structure to N-acetylmuramoyl-L-alanine amidases, such as T7 lysozyme, and some PGRPs are similarly catalytic, whereas others lack a critical cysteine residue in the catalytic triad (7). Thus, these PGRPs lack catalytic activity but function instead as pattern recognition receptors and/or as antimicrobials. For example, murine PGRP-S is directly antimicrobial and contributes to the neutrophil-mediated killing of bacteria (8, 9), whereas PGRP-SA, -SD, -LC, and -LE are key pattern recognition receptors involved in activation of the Drosophila immune response through the Toll or IMD (immune deficiency) signaling pathway (10 -16).In particular, genetic studies revealed that circulating PGRP-SA and PGRP-SD detect Gram-positive bacteria and activate the Toll pa...

Insects mount a robust innate immune response against a wide array of microbial pathogens. The hallmark of the Drosophila humoral immune response is the rapid production of antimicrobial peptides in the fat body and their release into the circulation. Two recognition and signaling cascades regulate expression of these antimicrobial peptide genes. The Toll pathway is activated by fungal and many Gram-positive bacterial infections, whereas the immune deficiency (IMD) pathway responds to Gram-negative bacteria. Recent work has shown that the intensity and duration of the Drosophila immune response is tightly regulated. As in mammals, hyperactivated immune responses are detrimental, and the proper down-modulation of immunity is critical for protective immunity and health. In order to keep the immune response properly modulated, the Toll and IMD pathways are controlled at multiple levels by a series of negative regulators. In this review, we focus on recent advances identifying and characterizing the negative regulators of these pathways. [BMB reports 2008; 41(4): 267-277]Insects rely primarily on innate immune responses to fight pathogens and have developed multiple mechanisms to recognize and respond to infection. The insect and mammalian innate immune responses exhibit a great deal of evolutionary conservation. One of the best examples of this conservation was provided by the discovery of the Toll pathway as a key component of the Drosophila immune response and the subsequent identification of the mammalian Toll-like Receptors (TLRs). In addition, the insect immune response relies on evolutionarily conserved NF-κB signaling cascades for the control of immune-induced gene expression. The genetic, genomic, and molecular tools available for studying the immune response in the fruit fly Drosophila melanogaster make this a favorite model system (1-4). Drosophila use several distinct effector mechanisms for immune protection including clotting, melanization, encapsulation, cell-based phagocytosis, and the inducible production of a battery of antimicrobial peptides. This antimicrobial peptide response is critical for protection against many microbial pathogens. Two signaling pathways regulate the production of these antimicrobial peptides in Drosophila -the immune deficiency (IMD) and Toll pathways. Recent work has shown that the intensity and duration of immune response is tightly regulated in Drosophila. As in mammals, over-exuberant immune responses are detrimental, and the proper down modulation of immunity is critical for health. In this review, we focus on the negative regulation of the IMD and the Toll pathway.

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.

Stereochemically defined peptide scaffolds are convenient tools for studying near neighbor effects on the reactivity of functional amino acid sidechains. The present study utilizes stereochemically defined peptide helices to effectively demonstrate that aspartic acid is an efficient catalytic residue in the Amadori arrangement. The results emphasize the structural determinants of Schiff base and Amadori product formation in the final accumulation of glycated peptides.