The Imd pathway

One of the major pathways controlling the production of AMPs in the fruit fly is the Imd pathway (Figure 2), which is often compared to the mammalian TNFR (tumor necrosis factor receptor) pathway. The first component of the pathway was originally

characterized in 1995, when Lemaitre et al. identified imd, a mutant that was unable to induce the production of certain AMPs after a bacterial infection (Lemaitre et al., 1995a). The imd mutants, however, expressed the antifungal gene Drosomycin normally, implying the presence of another signaling pathway, which was later found to be the Toll pathway (Lemaitre et al., 1996). After the initial discovery of the imd mutant, the other components of the pathway started to unravel.

The Imd pathway is activated by DAP-type PGN (Kaneko et al., 2004; Leulier et al., 2003), which is recognized by the transmembrane receptor PGRP-LC and by the intracellular PGRP-LE. PGRP-LC is the major pattern recognition receptor for the Imd pathway and signals the presence of an infection (Choe et al., 2002; Gottar et al., 2002; Rämet et al., 2002b). PGRP-LC is alternatively spliced to produce three different isoforms: PGRP-LCx, PGRP-LCa and PGRP-LCy, which have similar transmembrane domains and intracellular signaling domains, but differ in their extracellular PGRP domains, which have different binding specificities (Werner et al., 2000; Werner et al., 2003). PGRP-LCx binds polymeric DAP-type PGN, whereas LCa is not able to directly bind PGNs, but acts as a coreceptor for PGRP-LCx in the binding of the monomeric DAP-type PGN, called tracheal cytotoxin (TCT) (Chang et al., 2005; Chang et al., 2006; Mellroth et al., 2005). In addition to PGRP-LC, PGRP-LE acts as a receptor for DAP-type PGN in the Imd pathway, and it can be expressed both intra- and extracellularly. The cytosolic PGRP-LE is thought to be involved in the recognition of the PGNs of intracellular bacteria, such as Listeria monocytogenes, whereas the secreted form of PGRP-LE acts in collaboration with PGRP-LC in the recognition of extracellular PGNs (Kaneko et al., 2006; Neyen et al., 2012; Takehana et al., 2002; Takehana et al., 2004). Besides regulating Imd signaling, PGRP-LE is involved in the activation of the autophagy of intracellular bacteria (Yano et al., 2008).

The binding of bacterial DAP-type PGN to PGRP-LC leads to the dimerization of the receptor and the recruitment of the death-domain containing protein Imd, which shares homology with the mammalian RIP1 (receptor interacting protein) (Georgel et al., 2001). Imd interacts with the adaptor protein FADD (Fas-associated death domain) (Leulier et al., 2002; Naitza et al., 2002), which in turn binds and recruits the caspase-8 homolog Dredd (death-related Ced-3/Nedd2-like protein) to the signaling complex. Dredd is activated by ubiquitination by the ubiquitin E3 ligase Iap2 (Inhibitor of apoptosis 2) (Elrod-Erickson et al., 2000; Meinander et al., 2012).

After activation, Dredd cleaves Imd and exposes a binding site for Iap2, which leads to the K63-polyubiquitination of Imd (Paquette et al., 2010). Most likely, the next

Figure 2. Schematic representation of the Drosophila Imd pathway. Some components have been omitted for clarity. Key= Kenny, Ubi= ubiquitination, P= phosphorylation

step in Imd signaling is the recruitment of the Drosophila homolog of the mammalian MAPK kinase kinase TAK1 (TGF-β-activated kinase) and its adaptor protein TAB2 (TAK-binding protein 2) (Kleino et al., 2005; Silverman et al., 2003; Vidal et al., 2001;

Zhuang et al., 2006). The TAK1/TAB2 complex is involved in the phosphorylation of the Drosophila IKK (IκB kinase) complex, comprising of Kenny and Ird5 that are responsible of the phosphorylation of the NF-κB transcription factor Relish after it has been activated by Dredd through endoproteolytic cleavage (Kim et al., 2014;

Silverman et al., 2000; Stöven et al., 2003). Once the C-terminal inhibitory ankyrin-repeat domain of Relish (Rel-49) has been cleaved off, the N-terminal domain of Relish (Rel-68) translocates to the nucleus, where it induces the expression of AMPs and other target genes (Stöven et al., 2000; Wiklund et al., 2009). Similar to the mammalian NF-κB pathways, the Imd pathway branches into the JNK pathway downstream of TAK1/TAB2 and is involved in the control of the early response and the induction of genes participating in cytoskeletal remodeling and stress responses (Boutros et al., 2002; Rämet et al., 2002a; Valanne et al., 2007).

Imd signaling is tightly regulated at several levels, which adds complexity to the signaling pathway. Various components of the pathway are subject to posttranslational, hormonal and negative regulation. A negative regulator of the Imd pathway, Pirk, is rapidly induced in response to an infection. This creates a negative feedback loop, which functions likely at the level of the PRGRP-LC/Imd/FADD signaling complex (Aggarwal et al., 2008; Kleino et al., 2008; Lhocine et al., 2008).

The transcription factor Zfh1 has also been reported to function as a negative regulator of Imd signaling in vitro, but its role in vivo is less clear (Myllymäki and Rämet, 2013). Negative regulation of the Imd pathway occurs also at the level of PGRP-LC. PGRPs with an amidase activity, namely PGRP-LB, PGRP-SC1 and PGRP-SC2 downregulate the Imd pathway by digesting PGN into smaller fragments that have a decreased immunostimulatory activity and thereby reduce the activation of the pathway (Paredes et al., 2011; Zaidman-Remy et al., 2011). In addition to the amidase PGRPs, PGRP-LF can negatively regulate Imd signaling most likely by binding to PGRP-LC and by preventing its dimerization and hence, signal transduction (Basbous et al., 2011; Persson et al., 2007). In addition to the pathway being subject to negative regulation, Imd target genes are also transcriptionally controled in the fruit fly. In 2008, a Drosophila RNAi screen identified a previously unknown regulator of Imd signaling called Akirin, which acts downstream of Relish, but controls the expression of only a subset of Relish-dependent target genes (Bonnay et al., 2014; Goto et al., 2008). Recently, also ubiquitination and SUMOylation have been studied in the context of Imd signaling and they are now

known to be involved in the posttranslational modification of several Imd pathway components leading to either the activation or deactivation of the signaling cascade (Fukuyama et al., 2013; Kim et al., 2006; Meinander et al., 2012; Myllymäki et al., 2014; Thevenon et al., 2009; Tsuda et al., 2005).