The antimicrobial defence of Drosophila relies largely on the challenge-induced synthesis of an array of potent antimicrobial peptides by the fat body. The defence against Gram-positive bacteria and natural fungal infections is mediated by the Toll signalling pathway, whereas defence against Gram-negative bacteria is dependent on the Immune deficiency (IMD) pathway. Loss-of-function mutations in either pathway reduce the resistance to corresponding infections. The link between microbial infections and activation of these two pathways has remained elusive. The Toll pathway is activated by Gram-positive bacteria through a circulating Peptidoglycan recognition protein (PGRP-SA). PGRPs appear to be highly conserved from insects to mammals, and the Drosophila genome contains 13 members. Here we report a mutation in a gene coding for a putative transmembrane protein, PGRP-LC, which reduces survival to Gram-negative sepsis but has no effect on the response to Gram-positive bacteria or natural fungal infections. By genetic epistasis, we demonstrate that PGRP-LC acts upstream of the imd gene. The data on PGRP-SA with respect to the response to Gram-positive infections, together with the present report, indicate that the PGRP family has a principal role in sensing microbial infections in Drosophila.

The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. Here, we report that GNBP3 is a pattern recognition receptor that is required for the detection of fungal cell wall components. Strikingly, we find that there is a second, parallel pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host.

The Toll-dependent defense against Gram-positive bacterial infections in Drosophila is mediated through the peptidoglycan recognition protein SA (PGRP-SA). A mutation termed osiris disrupts the Gram-negative binding protein 1 (GNBP1) gene and leads to compromised survival of mutant flies after Gram-positive infections, but not after fungal or Gram-negative bacterial challenge. Our results demonstrate that GNBP1 and PGRP-SA can jointly activate the Toll pathway. The potential for a combination of distinct proteins to mediate detection of infectious nonself in the fly will refine the concept of pattern recognition in insects.

Ubiquitin proteases remove ubiquitin monomers or polymers to modify the stability or activity of proteins and thereby serve as key regulators of signal transduction. Here, we describe the function of the Drosophila ubiquitin-specific protease 36 (dUSP36) in negative regulation of the immune deficiency (IMD) pathway controlled by the IMD protein. Overexpression of catalytically active dUSP36 ubiquitin protease suppresses fly immunity against Gram-negative pathogens. Conversely, silencing dUsp36 provokes IMD-dependent constitutive activation of IMD-downstream Jun kinase and NF-kappaB signaling pathways but not of the Toll pathway. This deregulation is lost in axenic flies, indicating that dUSP36 prevents constitutive immune signal activation by commensal bacteria. dUSP36 interacts with IMD and prevents K63-polyubiquitinated IMD accumulation while promoting IMD degradation in vivo. Blocking the proteasome in dUsp36-expressing S2 cells increases K48-polyubiquitinated IMD and prevents its degradation. Our findings identify dUSP36 as a repressor whose IMD deubiquitination activity prevents nonspecific activation of innate immune signaling.

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The intracellular signaling pathway by which tumor necrosis factor (TNF) induces its pleiotropic actions is well characterized and includes unique components as well as modules shared with other signaling pathways. In addition to the currently known key effectors, further molecules may however modulate the biological response to TNF. In our attempt to characterize novel regulators of the TNF signaling cascade, we have identified transmembrane protein 9B (TMEM9B, c11orf15) as an important component of TNF signaling and a module shared with the interleukin 1␤ (IL-1␤) and Toll-like receptor (TLR) pathways. TMEM9B is a glycosylated protein localized in membranes of the lysosome and partially in early endosomes. The expression of TMEM9B is required for the production of proinflammatory cytokines induced by TNF, IL-1␤, and TLR ligands but not for apoptotic cell death triggered by TNF or Fas ligand. TMEM9B is essential in TNF activation of both the NF-B and MAPK pathways. It acts downstream of RIP1 and upstream of the MAPK and IB kinases at the level of the TAK1 complex. These findings indicate that TMEM9B is a key component of inflammatory signaling pathways and suggest that endosomal or lysosomal compartments regulate these pathways. Tumor necrosis factor (TNF)2 is a pleiotropic mediator of a wide range of cellular responses to infection, such as cytokine and chemokine production, cell migration, cell death, and cell differentiation and maturation (1). TNF plays a pivotal role in several autoimmune disorders such as rheumatoid arthritis; this is underscored by the clinical success of neutralizing TNF with antibodies or soluble receptors (2). A better understanding of intracellular TNF signaling is therefore of high clinical relevance.The two TNF receptors, TNFR1 (p55, TNFRSF1A) and TNFR2 (p75, TNFRSF1B), show high homology in their extracellular domains but less in their intracellular domains. Although soluble TNF binds TNFR1 with higher affinity than TNFR2 and therefore acts primarily via TNFR1, membranebound TNF activates equally TNFR1 and TNFR2 (3). In most tissues TNF signaling is mediated by TNFR1, whereas TNFR2 is restricted to fewer specific tissues, mostly of an immunological nature (3). Upon ligand binding, TNFR1 trimerizes and recruits TNF receptor-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP1), and TNF receptor-associated factor 2 (TRAF2). This first complex acts as a platform at the plasma membrane to activate the NF-B and MAPK signaling cascades, promoting cell survival and the expression of inflammatory cytokines. In a second step, TNFR1 is internalized into endocytic vesicles together with TRADD and RIP1 and recruits the proapoptotic molecules Fas-associated death domain (FADD) and caspase-8. This complex will initiate the apoptotic cell death program if concurrent anti-apoptotic NF-B activation is absent (4).The TGF␤-activated kinase 1 (TAK1) complex has a central function in many inflammatory pathways such as the TNFR, interleukin 1␤ receptor (IL-1R), and several Toll-...

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