Pattern recognition receptors in Drosophila immune responses

Developmental and Comparative Immunology 102 (2020) 103468

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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/devcompimm

Pattern recognition receptors in Drosophila immune responses a,b

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Yuzhen Lu , Fanghua Su , Qilin Li , Jie Zhang , Yanjun Li , Ting Tang , Qihao Hu , ⁎ Xiao-Qiang Yua,b, a

Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China

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Keywords: Pattern recognition receptors Pathogen-associated molecular patterns Innate immunity Drosophila

Insects, which lack the adaptive immune system, have developed sophisticated innate immune system consisting of humoral and cellular immune responses to defend against invading microorganisms. Non-self recognition of microbes is the front line of the innate immune system. Repertoires of pattern recognition receptors (PRRs) recognize the conserved pathogen-associated molecular patterns (PAMPs) present in microbes, such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA) and β-1, 3-glucans, and induce innate immune responses. In this review, we summarize current knowledge of the structure, classification and roles of PRRs in innate immunity of the model organism Drosophila melanogaster, focusing mainly on the peptidoglycan recognition proteins (PGRPs), Gram-negative bacteria-binding proteins (GNBPs), scavenger receptors (SRs), thioester-containing proteins (TEPs), and lectins.

1. Introduction All multicellular eukaryotes live in diverse environments with a variety of microbial pathogens, and the survival of hosts depends on the ability to identify and fight foreign microbes. Pattern recognition receptors (PRRs) have been found to play a crucial role in pathogen recognition by sensing conserved microbial structures designated as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA) and fungal β-1, 3-glucans (Akira et al., 2006). The non-self recognition of microbes is fundamental and central to trigger the subsequent immune responses. The innate immune system of insects is mainly composed of humoral and cellular immune responses (Lemaitre and Hoffmann, 2007). Humoral responses are characterized by induction of antimicrobial peptides (AMPs) regulated by the Toll and Imd (immune deficiency) pathways, and melanization of the invading microbes through the prophenoloxidase (PPO) activating system (Lu et al., 2014; Yi et al., 2014). Cellular responses are best illustrated by phagocytosis and nodulation of microbes (Strand, 2008). The fruit fly Drosophila melanogaster is a well-established genetic system for studying the physiology, immunity and pathology, and thus serves as an effective model to study PRRs. In this review, we discuss

the role of Drosophila PRRs in microbial recognition. We focus on the structure and function of peptidoglycan recognition proteins (PGRPs) and Gram-negative bacteria-binding proteins (GNBPs) that are mainly involved in initiation of signaling pathways, as well as scavenger receptors (SRs), thioester-containing proteins (TEPs) and lectins that promote the attachment of microbes to phagocytic hemocytes (Table 1). 2. Peptidoglycan-recognition proteins (PGRPs) Peptidoglycan-recognition proteins (PGRPs) are most versatile PRRs present in most invertebrates and mammals, but not in lower metazoan or plants. All PGRPs contain at least one conserved PGRP domain, which is homologous to bacteriophage and bacterial type 2 amidases (Wang et al., 2018a). PGRPs can be divided into short (S) and long (L) forms based on their sizes. Short PGRPs are mainly extracellular proteins of less than 20 kDa, while long PGRPs are usually double size of the short PGRPs with a variable N-terminal sequence, and they can be extracellular (such as PGRP-LBpc), transmembrane (such as PGRP-LC) or intracellular proteins (such as PGRP-LEfl and PGRP-LBpa) (Charroux et al., 2018; Werner et al., 2000). PGRPs can also be divided into catalytic PGRPs and non-catalytic PGRPs depending on their amidase

⁎ Corresponding author. Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China. E-mail address: [email protected] (X.-Q. Yu).

https://doi.org/10.1016/j.dci.2019.103468 Received 16 July 2019; Received in revised form 7 August 2019; Accepted 16 August 2019 Available online 17 August 2019 0145-305X/ © 2019 Elsevier Ltd. All rights reserved.

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Table 1 Pattern recognition receptors in Drosophila. Gene

Putative Ligand

Evidence

Refs.

PGRP-LC

DAP-type PGN, G- bacteria

Activate Imd pathway in vivo; rPGRP-LCx overexpressing flies reduce in immune response due to endosomal degradation of receptors.

PGRP-LE

DAP-type PGN, G- bacteria, L. monocytogenes

PGRP-SA

Lys-type PGN, G+ bacteria DAP-type PGN, G- bacteria, G+ bacteria

Help PGRP-LC recognize PGN; overexpression and null mutants activate PPO cascade; overexpression induce autophagy to eliminate L. monocytogenes. Mutants are susceptible to G+ bacteria; mutants defect in activation of the Toll pathway and phagocytosis of S. aureus. Form complex with GNBP1 and PGRP-SA to activate the Toll pathway; mutants defect in activation of the Imd pathway and increase susceptibility to DAP-type bacteria RNAi flies show that PGRP-LB downregulates the Imd pathway upon Gbacterial infection; PGRP-LBPC overexpression prevents the Imd pathway activation by gut endosymbiont. Null mutants up-regulate the Imd signaling and reduce activation of the Toll signaling; PGRP-SC1a mutant defects in phagocytosis of S. aureus; PGRP-SC1b hydrolyzes PGN. Null mutants up-regulate the Imd signaling and decrease activation of the Toll signaling; over-expression reduces gut commensal dysbiosis. Degrades PGN and shows antibacterial activity to Bacillus. Interacts with PGRP-LCx to block the Imd pathway; null mutants inhibit apoptosis in epidermal cells. Overexpression activates the Imd pathway. Binds to LPS and β-1, 3-glucan; hydrolyzes Lys-PGN; interacts with PGRP-SA and PGRP-SD to activate the Toll pathway.

Choe et al. (2002); Gottar et al. (2002); Kaneko et al. (2004); Neyen et al. (2016) Kaneko et al. (2006); Takehana et al. (2002); Yano et al. (2008)

PGRP-SD

PGRP-LB

DAP-type PGN, G- bacteria

PGRP-SC1

PGN, possibly G+ bacteria, G- bacteria

PGRP-SC2

Possibly G- bacteria, G+ bacteria DAP-type PGN, Bacillus –

PGRP-SB1 PGRP-LF PGRP-LA GNBP1

E. carotovora LPS, β-1,3-glucan, Lys-PGN, certain G+ bacteria

GNBP3

β-1,3-glucan, polysaccharides, fungi

dSR-CI Peste

certain G+ and G- bacteria Mycobacteria, intracellular bacteria

Croquemort

certain G+ and G- bacteria, fungi

Draper

LTA, E. coli, S. aureus Possibly G+ and cell envelop disrupted G- bacteria, Zygomycetes

Eater

NimC1

Possibly G+ and G- bacteria

TEPs

TEP6

Possibly G+ bacteria, fungi, wasp E. coli, P. gingivalis, Photorhabdus S. aureus, H. bacteriophora Photorhabdus, P. gingivalis, P. aeruginosa C. albicans, Photorhabdus

DSCAM

Possibly E. coli

Lectin

E. coli, E. chrysanthemi

TEP2 TEP3 TEP4

Binds to long chain β-1, 3-glucan and agglutinates fungal spores; overexpression activates the Toll pathway; GNBP3 mutant reduces in PO activity. RNAi in S2 cells reduces phagocytosis of E. coli and S. aureus. RNAi in S2 cells reduces phagocytosis of Mycobacteria and the entry of intracellular bacteria. Null flies are susceptible to certain bacteria and fungi infection; RNAi flies are susceptible to L. amazonensis. RNAi in S2 cells and null mutant flies reduce phagocytosis of E. coli and S. aureus. Binds to G+ bacteria and cell envelop disrupted G- bacteria; null hemocytes are impaired in phagocytosis of certain G+ bacteria; null mutants are susceptible to S. marcescens and Zygomycetes infection. Binds to certain heat-inactivated bacteria; null hemocytes reduce phagocytosis of latex beads, yeast particles. Tep1-2-3-4 quadruple mutants are sensitive to entomopathogenic fungi, G+ bacteria and parasitoid wasp. Null mutants reduce phagocytosis of E. coli; null mutants are susceptible to P. gingivalis, but resistant to Photorhabdus RNAi in S2 cells decrease phagocytosis of S. aureus; mutants are susceptible to H. bacteriophora infection Phagocytosis of P. aeruginosa is decreased in null mutant hemocytes; null mutants are susceptible to P. gingivalis and P. aeruginosa, but resistant to Photorhabdus Binds to and phagocytoses C. albicans; RNAi in S2 cells decreases phagocytosis of C. albicans; null mutants decrease phagocytosis of inactive E. coli; null mutants are resistant to Photorhabdus Binds to E. coli; RNAi hemocytes and S2 cells pre-incubated with antibody reduce phagocytosis of E. coli; Null hemocytes bind E. coli with equal efficiency as wild type. DL1 binds to E. coli and E. chrysanthemi; DL2 or DL3 binds to hemocytes and agglutinated E. coli.

activity (Paredes et al., 2011). The catalytic PGRPs contain three Zn2+coordinating residues (His, His, Cys) in the PGN binding groove, while the non-catalytic PGRPs lack the key cysteine residue for zinc binding and cannot hydrolyze PGN (Low et al., 2011). The catalytic PGRPs, such as PGRP-SB1, -SC1 and -LB, are mainly located extracellularly and act as modulators of the immune signal pathways by sequestering PGN released by bacteria. The non-catalytic PGRPs, including PGRP-SA, -LC and -LE, initiate signal transduction or regulate the activation of immune responses (Wang et al., 2019). PGRPs are able to recognize different types of PGN from various bacterial strains (Leulier et al., 2003) and initiate immune responses, including activation of the Toll, Imd and PPO pathways (Fig. 1) (Royet

Bischoff et al. (2004); Michel et al. (2001); Wang et al. (2006), 2008 Bischoff et al. (2004); Iatsenko et al. (2016); Leone et al. (2008); Wang et al. (2008) Charroux et al. (2018); Kim et al. (2000); Zaidman-Remy et al. (2006) Bischoff et al. (2004); Costechareyre et al. (2016); Garver et al. (2006); Mellroth et al. (2003) Costechareyre et al. (2016) Mellroth and Steiner (2006) Basbous et al. (2011); Maillet et al. (2008); Tavignot et al. (2017) Gendrin et al. (2013) Gobert et al. (2003); Kim et al. (2000); Pili-Floury et al. (2004); Wang et al. (2006), 2008 Fullaondo et al. (2011); Gottar et al. (2006); Matskevich et al. (2010); Mishima et al. (2009) Ramet et al. (2001) Agaisse et al. (2005); Philips et al. (2005) Guillou et al. (2016); Okuda et al. (2016); Stuart et al. (2005) Cuttell et al. (2008); Hashimoto et al. (2009) Bretscher et al. (2015); Chamilos et al. (2008); Chung and Kocks (2011); Kocks et al. (2005); Melcarne et al. (2018) Kurucz et al. (2007); Melcarne et al. (2018); Zsámboki et al. (2013) Dostalova et al. (2017) Igboin et al. (2011); Shokal et al. (2017); Stroschein-Stevenson et al. (2006) Arefin et al. (2014); Stroschein-Stevenson et al. (2006) Haller et al. (2018); Igboin et al. (2011); Shokal et al. (2017) Shokal and Eleftherianos (2017a); Shokal et al. (2017); Stroschein-Stevenson et al. (2006) Vlisidou et al. (2009); Watson et al. (2005)

Ao et al. (2007); Tanji et al. (2006)

et al., 2011; Wang et al., 2019). PGNs with a lysine residue in the 3rd amino acid of the PGN short peptide (Lys-type PGN), which are present in most Gram-positive bacteria, activate the Toll pathway through the cooperation of PGRP-SA, PGRP-SD and GNBP1 (Gram-negative bacteria-binding protein 1) (Bischoff et al., 2004; Michel et al., 2001; Wang et al., 2006, 2008). PGNs with the diaminopimelic acid in the corresponding position (DAP-type PGN), which are present in most Gramnegative bacteria and Gram-positive bacilli, elicit the Imd pathway through PGRP-LC (Gottar et al., 2002; Ramet et al., 2002). The transmembrane PGRP-LCx homodimer can recognize polymeric DAP-type PGN (Choe et al., 2002; Kaneko et al., 2004), whereas the PGRP-LCaPGRP-LCx heterodimer and intracellular full-length PGRP-LEfl can 2

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Fig. 1. Functions of pattern recognition receptors in Drosophila immune responses. (A) The roles of PGRPs in immune signaling pathways. Lys-type PGN from Grampositive bacteria is recognized by the PGRP-SA, PGRP-SD and GNBP1 complex. Fungal β-glucan is recognized by GNBP3. Virulence factors from bacteria and fungi are recognized by the protease Persephone. The recognition of these microbial molecules by PRRs induces a proteases cascade that finally cleaves pro-Spätzle-1 into an active ligand Spätzle-1 (Spz-1) for the Toll pathway. PGRP-SCs are required for fully activation of the Toll pathway. DAP-type PGNs from Gram-negative bacteria or Gram-positive bacilli are recognized by PGRP-LC, with monomeric PGN by PGRP-LCa-PGRP-LCx heterodimer and polymeric PGN by PGRP-LCx homodimer, to elicit the Imd pathway. Extracellular PGRP-LEpg facilitates the interaction of PGN with PGRP-LCx. PGRP-SD binds to DAP-type PGN and enhances the activation of the Imd pathway. PGRP-LF and PGRP-LCx can form non-signaling heterodimers to down-regulate the Imd pathway. rPGRP-LC binds to polymeric PGN and dampens immune activation via degradation of PGRP-LC. PGRP-LB and PGRP-SC have amidase activity and act as negative regulators of the Imd pathway by eliminating PGN. PGRP-SB1 directly degrades DAP-type PGN to kill bacteria. PGRP-LEfl enhances the clearance of intracellular bacteria by activating autophagy. Toll-7 interacts with virus to induce antiviral autophagy. (B) Pattern recognition receptors involved in the prophenoloxidase (PPO) pathway. PPO is cleaves into phenoloxidase (PO) by a cascade of serine proteases, PO oxidizes phenolic molecules to produce melanin. PGRP-LE, GNBP3, thioester-containing proteins (TEP2 and TEP4), C-type lectins (DL2 and DL3) can activate PPO pathway. (C) Pattern recognition receptors in phagocytosis and agglutination. Class C scavenger receptor (dSR-CI), CD36 family proteins (Croquemort and Peste), Nimrod family proteins (Draper, Eater and NimC1), and Dscam are transmembrane pattern recognition receptors that mediate phagocytosis. PGRP-SC1a, thioester-containing proteins (TEP2, TEP3, TEP4 and TEP6) are secreted proteins that enhance phagocytosis. The box represents C-type lectins that participate in bacterial agglutination in Drosophila.

pathway and provides a negative feedback regulation to immune responses (Zaidman-Remy et al., 2006). The secreted PGRP-LB hydrolyzes PGN in gut to prevent the diffusion of PGN, while the transmembrane and cytosolic PGRP-LB isoforms cleave monomeric PGN to prevent endosymbiont-induced PGRP-LC-dependent immune responses (Charroux et al., 2018; Paredes et al., 2011; Zaidman-Remy et al., 2006). PGRP-SC1a/b and PGRP-SC2 are responsible for the removal of peptides from the glycan chains and negatively regulate the Imd pathway synergized with PGRP-LB and Pirk (Mellroth et al., 2003; Paredes et al., 2011). In addition, PGRP-SC1 and PGRP-SC2 are also required for fully activation of the Toll pathway in flies (Costechareyre et al., 2016; Garver et al., 2006). Drosophila PGRPs also participate in other antibacterial mechanism. PGRP-LE can bind PGN to activate the PPO cascade (Fig. 1B) (Takehana et al., 2002, 2004). The full-length PGRP-LEfl can also induce autophagy to eliminate intracellular bacteria such as Listeria monocytogenes (Yano et al., 2008). PGRP-SB1 can act as bactericide through enzymatic degradation of DAP-type PGN (Mellroth and Steiner, 2006). PGRP-SC1 is able to mediate phagocytosis of Gram-positive Staphylococcus aureus (Garver et al., 2006). More details on PGRPs can be found in other reviews (Kurata, 2014; Royet et al., 2011; Wang et al., 2018a, 2018b, 2019).

recognize monomeric PGN (Fig. 1A) (Kaneko et al., 2004, 2006). PGRPs can modulate immune responses by binding to PGN or transmembrane receptors (Fig. 1A). Extracellular PGRP-LEpg (a cleaved form of PGRP-LE) stimulates the Imd signaling pathway by facilitating the interaction of PGN with PGRP-LCx (Kaneko et al., 2006; Takehana et al., 2002, 2004). Although PGRP-SD mutants are susceptible to Gram-positive bacteria, PGRP-SD binds DAP-type PGN and enhances activation of the Imd pathway in response to both polymeric and monomeric PGN. PGRP-SD interacts with PGRP-LCx only in the presence of polymeric PGN (Iatsenko et al., 2016; Leone et al., 2008). PGRP-LA is not required for systemic immune responses but participates in the activation of the Imd pathway in barrier epithelia (Gendrin et al., 2013). PGRP-LF, rPGPR-LC (an alternative splice variant of PGRP-LC), PGRP-LB, PGRP-SC1a/b and PGRP-SC2 are negative regulators of the Imd pathway (Fig. 1A) (Paredes et al., 2011; Wang et al., 2019). PGRPLF competes with PGRP-LCa for interaction with PGRP-LCx and forms non-signaling heterodimers to down-regulate the Imd pathway (Basbous et al., 2011; Maillet et al., 2008). The negative regulation of the Imd pathway by PGRP-LF is crucial for proper induction of apoptosis in flies (Tavignot et al., 2017). rPGPR-LC selectively responds to polymeric PGN from dead bacteria and dampens immune activation via rPGRP-LC-mediated endocytosis and ESCRT-dependent degradation of PGRP-LC (Neyen et al., 2016). PGRP-LB is regulated by the Imd 3

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3. Gram-negative bacteria-binding proteins/β-glucan recognition proteins (βGRPs)

embryonic development (Pearson et al., 1995), and acts as a pattern recognition molecule to help phagocytose both Escherichia coli and S. aureus but not yeast (Ramet et al., 2001). SR-CIII and SR-CIV RNAi flies are susceptible to Leishmania amazonensis infection (Fig. 1C) (Okuda et al., 2016).

Gram-negative bacteria-binding proteins (GNBPs)/β-glucan recognition proteins (βGRPs) belong to the β-1, 3-glucanase protein family and include several proteins involved in innate immune recognition (Hughes, 2012). Three GNBPs are encoded in the Drosophila genome (Kim et al., 2000). GNBP1 is expressed throughout Drosophila life stages, but GNBP2 and GNBP3 show weak signals during embryogenesis (Kim et al., 2000). GNBP1 has a high affinity for LPS and β-1, 3-glucan but not for PGN, β-1, 4-glucan, or chitin (Kim et al., 2000). The expression of AMP genes activated by LPS or β-1, 3-glucan is enhanced in cells overexpressing GNBP1 and inhibited by blocking GNBP1, suggesting a role of GNBP1 in recognition of Gram-negative bacteria and fungi (Kim et al., 2000). A survey on statistical associations between bacterial load and genotypes shows that polymorphism in the GNBP locus, consisting of GNBP1 and GNBP2 genes, is significantly associated with resistance to Gram-negative bacteria (Sackton et al., 2010). However, GNBP1 mutant leads to compromised survival after infections by some Gram-positive bacteria but not fungal or Gram-negative bacterial challenge (Gobert et al., 2003; Pili-Floury et al., 2004). These Gram-positive bacteria activate immune responses in a PGRP-SA- and GNBP1-independent manner (Bischoff et al., 2004). GNBP1 is able to hydrolyze Lys-type PGN, promoting the binding of PGRP-SA to PGN fragments (Filipe et al., 2005; Wang et al., 2006). PGRP-SD enhances the binding of GNBP1 to Lystype PGN and stabilizes the interaction between GNBP1 and PGRP-SA by binding to GNBP1 (Wang et al., 2008). Thus, GNBP1, PGRP-SA and PGRP-SD act in concert to activate the Toll pathway in defense against Gram-positive bacteria (Fig. 1A). GNBP3 binds to β-1, 3-glucan of the fungal cell wall and participates in defense against fungal infections (Fig. 1A) (Gottar et al., 2006). An occluding loop in the N-terminus of GNBP3 is essential for discriminating short and long polysaccharides and binds to long chain β-1, 3-glucan (Mishima et al., 2009). GNBP3 functions upstream of the Modular serine protease (ModSP) or an unknown protease that is inhibited by serpin Spn1 to activate the Toll pathway during fungal infection (Fullaondo et al., 2011; Gottar et al., 2006). GNBP3 also agglutinates fungi and interacts with serpin Necrotic and PPO to activate melanization in a Toll-independent manner (Fig. 1B) (Matskevich et al., 2010).

4.2. CD36 family CD36 scavenger receptors are transmembrane and highly glycosylated glycoproteins that belong to Class B scavenger receptors, and they play vital roles in lipid metabolism and innate immunity (Canton et al., 2013). Croquemort (Crq), one of the CD36 family SRs encoded in Drosophila, is expressed on plasmatocytes and required for phagocytes to efficiently uptake a broad range of bacteria and fungi (Guillou et al., 2016; Stuart et al., 2005). Croquemort acts parallel to the Toll and Imd pathways to eliminate microbes with a role in bacteria phagocytosis and phagosome maturation (Guillou et al., 2016). Also, Croquemort is essential for efficient phagocytosis of apoptotic corpses (Franc et al., 1999). RNAi flies of Croquemort and two other CD36-like receptors, namely CG10345 and CG31741, are susceptible to L. amazonensis infection (Okuda et al., 2016). Peste, another CD36 family member, plays a role in the recognition and uptake of Mycobacterium and L. amazonensis, but not E. coli or S. aureus by S2 cells (Fig. 1C) (Agaisse et al., 2005; Philips et al., 2005). 4.3. Nimrod family Nimrod proteins are characterized by the presence of NIM repeats, a special type of the epidermal growth factor (EGF) domain presented in mammalian class F SRs. The Nimrod proteins can be divided into three types based on the structural features: Draper-type (NimA, Draper), Nimrod B-type (NimB) and Nimrod C-type (NimC, Eater) proteins (Somogyi et al., 2008). Ten Nimrod related genes cluster in the chromosome 2, which encode the transmembrane protein NimA with a single NIM domain followed by variable numbers of EGF domains, the secreted proteins NimB1-5 with 1–8 NIM domains, and the transmembrane proteins NimC1-4 with 2–16 NIM domains. Genes encoding Draper with a NIM domain followed by 15 EGF domains and Eater with 32 NIM repeats are not located in the Nimrod gene cluster (Kurucz et al., 2007). Draper, Eater and NimC1 are best characterized Nimrod receptors participated in pathogen phagocytosis.

4. Scavenger receptors (SRs)

4.3.1. Draper Draper is expressed in glia, hemocytes and other tissues, and it is involved in the phagocytosis of apoptotic cells, neuronal axons, degenerating dendrites, salivary gland cells, germline cells and bacteria (E. coli and S. aureus) (Cuttell et al., 2008; Hilu-Dadia et al., 2018; Shiratsuchi et al., 2012). The extracellular region of Draper binds lipoteichoic acid and mediates phagocytosis of S. aureus (Hashimoto et al., 2009). Draper and integrin βν, a receptor in the phagocytosis of apoptotic cells, can cooperatively enhance the phagocytic elimination of S. aureus by recognizing distinct cell wall components (Shiratsuchi et al., 2012). The integrin α-subunit αPS3 forms a complex with βν to mediate the phagocytosis of apoptotic cells and bacteria (Nonaka et al., 2013).

Scavenger receptors are a large family of multi-domain receptors that carry out multiple functions in physiological or pathological processes, such as pathogen clearance, lipid transport, and cargo transport within the cell (Canton et al., 2013). Mammalian SRs have been reported to recognize a large repertoire of ligands, including modified or endogenous molecules derived from the host and PAMPs such as LPS, LTA, bacterial CpG DNA, β-1, 3-glucan and viral products (Areschoug and Gordon, 2009; MacLeod et al., 2015). SRs are grouped into nine heterogeneous classes (A-I, but class C is absent in mammals) (Canton et al., 2013; PrabhuDas et al., 2017), and only a few SRs have been identified as PRRs in Drosophila (Table 1) (Khush and Lemaitre, 2000; Pearson et al., 1995).

4.3.2. Eater The Eater transmembrane receptor is expressed predominantly in Drosophila plasmatocytes and its transcription is regulated by a GATA factor Serpent (Kroeger et al., 2012). Eater is required for sessility and adhesion in hemocytes (Bretscher et al., 2015). Eater null flies show impaired phagocytosis of S. aureus, E. coli and Serratia marcescens, and display decreased survival against S. marcescens infection (Kocks et al., 2005). However, hemocytes from Eater null flies show defects in phagocytosis of Gram-positive bacteria (S. aureus, Staphylococcus epidermidis and Micrococcus luteus), but not Gram-negative bacteria (E. coli

4.1. Class C SRs The class C SRs are first identified in Drosophila with broad polyanionic ligand-binding specificity similar to that of mammalian SR-A (Pearson et al., 1995). Four class C SRs (SR-CI, -CII, -CIII and -CIV) have been identified in Drosophila and they exhibit different evolutionary trajectories. SR-CI, -CIII and -CIV evolve rapidly and show high levels of polymorphism, while SR-CII is evolutionarily conserved (Lazzaro, 2005). Drosophila SR-CI is expressed only in hemocytes during 4

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2011). Tep2-3 double mutant or Tep2-3-4 triple mutant flies are resistant to different microbes (Bou Aoun et al., 2011). Tep2, Tep3 or Tep6 RNAi S2 cells show decreased ability in phagocytosis of E. coli, S. aureus or Candida albicans, respectively (Stroschein-Stevenson et al., 2006). Tep3 mutants are susceptible to entomopathogenic nematodes Heterorhabditis bacteriophora infection (Arefin et al., 2014). Both Tep2 and Tep4 null mutants are susceptible to Pseudomonas gingivalis (Igboin et al., 2011). TEP4 is required for phagocytosis and host defense against P. aeruginosa; however, TEP4-mediated opsonization can be eluded by P. aeruginosa quorum-sensing regulator RhlR (Haller et al., 2018). Drosophila TEPs also act as regulators and effectors against the entomopathogenic bacteria Photorhabdus. TEP2, TEP4 and TEP6 loss of function mutants are resistant to Photorhabdus asymbiotica or P. luminescens (Shokal and Eleftherianos, 2017a, 2017c; Shokal et al., 2017). TEP2 and TEP4 participates in energy reserves, inflammation, and phenoloxidase activation against Photorhabdus infection (Shokal and Eleftherianos, 2017c; Shokal et al., 2017, 2018). TEP2, TEP4 and TEP6 differently influence the activation of Toll, Imd, JAK/STAT and JNK signaling pathways during Photorhabdus infection (Shokal and Eleftherianos, 2017c; Shokal et al., 2017). Recent studies show that Tep1-2-3-4 quadruple mutants are sensitive to entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae), Gram-positive bacteria (S. aureus and E. faecalis) and parasitoid wasps (Asobara tabida and Leptopilina boulardi) (Dostalova et al., 2017). TEP1-4 are also involved in phagocytosis of E. faecalis and S. aureus, and function upstream or independently of the serine protease ModSP to activate the Toll pathway during infection by Gram-positive bacteria and entomopathogenic fungi (Dostalova et al., 2017). These studies suggest that TEPs have both specific and redundant functions in pathogen recognition and immune responses (Table 1).

and S. marcescens) (Bretscher et al., 2015; Melcarne et al., 2018). Another study shows that the survival of Eater mutant flies decreases upon infection by M. luteus and Enterococcus faecalis but not by S. aureus due to defective phagocytosis (Nehme et al., 2011). Eater neither plays a role in immune responses to infection by Gram-negative Porphyromonas gingivalis (Igboin et al., 2011). These studies indicate that Eater may act as the main receptor for phagocytosis of Gram-positive but not Gramnegative bacteria (Bretscher et al., 2015; Melcarne et al., 2018). Further studies show that recombinant Easter can directly target live Grampositive bacteria but not live Gram-negative bacteria (E. coli, S. marcescens and Pseudomonas aeruginosa). However, Eater can bind to Gramnegative bacteria with disrupted cell envelope resulting from treatments with antimicrobial peptides or lysozymes (Chung and Kocks, 2011, 2012). In addition, Eater null flies display increased susceptibility to Rhizopus oryzae infection, indicating that Eater also participates in immune responses against Zygomycetes infection (Chamilos et al., 2008). 4.3.3. NimC1 NimC1 is initially identified as the antigen of a hemocyte-specific antibody (P1) involved in phagocytosis of S. aureus and E. coli (Kurucz et al., 2007). Knockdown of NimC1 in plasmatocytes inhibits the phagocytosis of S. aureus, while overexpression of NimC1 in S2 cells stimulates the phagocytosis of both S. aureus and E. coli (Kurucz et al., 2007). Binding assays show that NimC1 does not bind to live bacteria, but binds to the heat-inactivated Gram-negative E. coli, S. marcescens, Xanthomonas campestris, P. aeruginosa and Gram-positive Bacillus cereus var. mycoides (Zsámboki et al., 2013). However, studies from NimC1 null hemocytes show that NimC1 is not required for ex vivo phagocytosis of both heat-inactivated Gram-positive and Gram-negative bacteria, but is essential for uptake of latex beads and zymosan yeast particles (Melcarne et al., 2018). Hemocytes from single and double null flies reveal that NimC1 and Eater act synergistically in phagocytosis of a broad range of bacteria, latex beads and yeast particles (Melcarne et al., 2018). NimC1 transcription is regulated by Jumeau, a winged-helix/forkhead (FKH) transcription factor required for hemocyte phagocytosis (Hao et al., 2018). NimC1 and Eater are both cell surface markers of plasmatocytes involved in hemocyte adhesion and negatively regulate hemocyte counts in larvae (Melcarne et al., 2018). Studies also show that NimA, NimB1 and NimB2 bind to certain bacteria with different affinity, and only NimB1 can bind to S. epidermidis (Zsámboki et al., 2013). NimC3 RNAi flies are susceptible to L. amazonensis infection (Okuda et al., 2016), and NimC4 (Simu) is required for efficient clearance of apoptotic cells (Kurant et al., 2008).

6. Down syndrome cell adhesion molecule (Dscam) The Down syndrome cell adhesion molecule (Dscam) gene encodes an immunoglobulin (Ig)-containing protein that is composed of 9(Ig)4(FN)-Ig-2(FN) (FN stands for fibronectin type III domain) and usually followed by a transmembrane domain and a cytoplasmic tail (Armitage and Brites, 2016). Secreted isoforms of Dscam without transmembrane domain and cytoplasmic tail are also detected in the hemolymph of Drosophila larvae (Watson et al., 2005). Dscam is mainly expressed in neurons, fat body cells and hemocytes with the potential to produce thousands of protein isoforms (Dscam hypervariable, Dscam-hv) due to mutually exclusive alternative splicing of the exons encoding half of the Ig2, half of the Ig3 and the whole Ig7 (Watson et al., 2005). Two alternative splicing exons encoding the transmembrane domain and exons regularly splicing for different cytoplasmic tails also contribute to the diversity of Dscam-hv (Armitage and Brites, 2016). Dscam-hv plays an vital role in nervous system wiring by homophilic binding between Dscam-hv isoforms for recognition of each other and promoting self-avoidance (Wojtowicz et al., 2007). In addition, the link between Dscam and the immune system has been the subject of a number of studies (Armitage et al., 2015; Armitage and Brites, 2016). Although growing evidence, especially studies in mosquito and shrimp, indicate that Dscam-hv may function as a pathogen-specific recognition molecule and be involved in immune memory that is analogous to vertebrate adaptive immunity, its role in invertebrate immunity remains controversial. The Drosophila immune-competent cells have the potential to express more than 18000 diverse Dscam isoforms based on the numbers of alternative exons that are detected in the fat body or hemocytes (Watson et al., 2005). Recombinant expressed Dscam proteins bind to E. coli, and hemocytes from Dscam knockdown larvae or S2 cells pre-incubated with Dscam antibody showed reduced phagocytosis activity against bacteria, suggesting that Dscam may act as a phagocytic receptor or an opsonin (Watson et al., 2005). However, unlike Anopheles gambiae Dscam, in which the diversity of Ig2 and Ig3 variants increased

5. Thioester-containing proteins (TEPs) In vertebrates, the complement system is an important component of innate immunity and eliminates foreign cells through an enzyme cascade, leading to pathogen recognition, opsonization and lysis (Bou Aoun et al., 2011). Thioester-containing proteins (TEPs) are conserved proteins among insects that show sequence similarities with both the complement factors C3/C4/C5 and the α2-macroglobulin family (Shokal and Eleftherianos, 2017b). Most TEPs contain a GCGEQ motif that allows covalent bond formation of TEPs with microbial surfaces to promote opsonization (Bou Aoun et al., 2011). Drosophila contains six Tep genes (Tep1-6), of which Tep5 may be a pseudogene with no expression and TEP6 (Macroglobulin complement related, Mcr) lacks a functional thioester motif (Lagueux et al., 2000; Shokal and Eleftherianos, 2017b). Drosophila Tep1-4 and Tep6 genes are induced in both larvae and adults upon bacterial, fungal, parasitoid and parasitic challenges (Fig. 1C) (Castillo et al., 2015; Lagueux et al., 2000; Shokal and Eleftherianos, 2017c; Shokal et al., 2017). Single Tep1, Tep2, Tep3 or Tep4 deficient adult flies are not significantly susceptible to a broad range of Gram-positive or Gram-negative bacteria (Bou Aoun et al., 5

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(Pace et al., 2002). However, galectins are not induced in Drosophila adult flies treated with mixed bacterial culture of S. marcescens and E. faecalis (Sackton et al., 2017), the function of Drosophila galectins as PRRs in innate immunity remains unclear.

after pathogen infection, the transcript diversity of Ig2 and Ig3 in Drosophila Dscam is not significantly affected after exposure to bacteria, virus and fungi at early stages (Armitage et al., 2014; Smith, 2012). Similarly, no short-term modulation of Drosophila Dscam gene expression is observed in hemocytes and fat body of larvae after exposure to E. coli, Pseudomonas fluorescens and Bacillus thuringiensis (Peuss et al., 2016). It has also been reported that embryonic hemocytes recognize and phagocytose E. coli in a Dscam-independent manner (Vlisidou et al., 2009). Thus, additional studies on host species, pathogen species, exposure time, Dscam diversity, and Dscam-mediated immune responses are required to verify the functions of Dscam in innate immunity.

8. Other pattern recognition receptors Drosophila Toll (Toll-1) receptor was first discovered to regulate dorso-ventral patterning in embryonic development and the subsequent studies reveal that the Toll (Toll-1-Spӓtzle-1) pathway also regulates expression of antimicrobial peptides in flies (Lemaitre and Hoffmann, 2007). Although Drosophila Toll-1 and mammalian Toll-like receptors (TLRs) regulate orthologous Toll pathways, Toll-1 and TLRs differ in the recognition process. Mammalian TLRs recognize and directly bind to different PAMPs, such as bacterial LPS, teichoic acid, PGN, CpG DNA and viral single-stranded and double-stranded RNAs; however, Drosophila Toll-1 binds to a cytokine like ligand Spӓtzle-1 (Spz-1) to trigger the signaling pathway (Kirk and Bazan, 2005). Also, Drosophila Toll-1 and Toll-7 can bind to Vesicular stomatitis virus (VSV) and activate promoter activity of several antimicrobial peptide genes (Chowdhury et al., 2019), and binding of Toll-7 with VSV can induce antiviral autophagy independently of the canonical Toll-1 pathway in adult flies (Nakamoto et al., 2012). Another study revealed that the autophagy gene Atg7 is required for full VSV resistance; however, the recognition of VSV is independent of Toll-7 (Lamiable et al., 2016). The serine protease Persephone (Psh) can mediate the Toll pathway when proteolytically activated by some bacterial or fungal proteases (El Chamy et al., 2008; Gottar et al., 2006). Activation of the Psh-dependent Toll pathway is decreased by microbes that are defect in proteolytic activity, suggesting that Persephone functions as an immune receptor to sense a broad range of microbial proteases that activate Psh through a bait region (Issa et al., 2018).

7. Lectins Lectins are carbohydrate-binding proteins containing at least one carbohydrate-recognition domain (CRD), and they can be classified into several families, including C-type lectins (CTLs), S-type lectins (galectins), I-type lectins, P-type lectins and pentraxins (Nilsson, 2011). In particular, CTLs and galectins can bind to the glycans of glycoproteins and glycolipids with high affinity and they play crucial roles in microbe recognition in invertebrates (reviewed in Adelman and Myles, 2018; Pees et al., 2016; Vasta et al., 2017; Xia et al., 2018). 7.1. C-type lectins C-type lectins/C-type lectin domain containing proteins (CTLDs) are a superfamily of calcium-dependent carbohydrate-binding proteins (Pees et al., 2016). The number of CTLs varies greatly in invertebrate species, from only 7–40 in insecta to 100–300 in nematoda and molluscs (Pees et al., 2016; Wang et al., 2018b; Xia et al., 2018). Most CTLs contain single CRD, while CTLs with 2–4 CRDs are also found in invertebrates (Wang et al., 2018b). Some CTLs contain additional domains that can carry out effector functions, such as chitin binding domain, complement control protein modules and coiled-coil domain (Sun et al., 2017). CTLs can bind to various microbe (such as bacteria, fungi, and viruses) or PAMPs (such as LPS, LTA, mannose, PGN), and they are involved in opsonization, nodule formation, agglutination, encapsulation, phagocytosis and even display bactericidal effect in invertebrates (Wang et al., 2018b; Xia et al., 2018). In addition, insect CTLs also participate in melanization, PPO activation and maintaining commensal homeostasis (Xia et al., 2018). Although there are more than 30 CTLs in flies, the functions of Drosophila CTLs are not well studied. Drosophila DL1, DL2 and DL3 are secreted galactose-specific lectins with similar hemagglutinating activities (Ao et al., 2007; Tanji et al., 2006). DL1 can bind to E. coli and Erwinia chrysanthemi, but studies in DL1 mutant flies show that it does not play a role in regulation of antibacterial peptide expression upon E. coli infection (Tanji et al., 2006). DL2 and DL3 bind to hemocytes and agglutinate E. coli but not S. aureus or Saccharomyces cerevisiae, and they may act as pattern recognition receptors to mediate encapsulation and melanization (Fig. 1C) (Ao et al., 2007).

9. Conclusions and future perspectives Non-self recognition is the fundamental step in insect defense against pathogens. Repertoires of PRRs are essential for immune response against a large range of pathogens. PGRPs are most well studied PRRs in flies and they play an important role in initiating and modulating signal transduction pathways, such as the Toll and Imd pathways, and the PPO cascade. The roles of PGRPs in gut immune homeostasis have been increasingly studied in recent years. Among the PGRPs, the role of PGRP-LA, -LCy, and -LD are still unclear. Dscam, CTLs, TEPs and Nimrod family proteins are diversified immune molecules, yet their functions in immunity are not well understood. Controversial results about functions of these proteins have been reported, suggesting that they may have specific or redundant roles against different pathogens. Many questions on the specific recognition and the molecular mechanism of PRRs remains unknown. For example, what is the relationship between the diversified PRRs and various microbes? What are the specific ligands for these PRRs except the wellknown PGRPs and GNBPs? How do PRRs, such as PGRP-LE and TEP2, activate the PPO cascade? The combination of the well-established forward genetic screens with multiomics, genetic manipulation, especially multi-gene operation by RNAi or CRISPR, can lead to a better knowledge of these recognition molecules and the underlying molecular mechanisms in modulation of immune responses, and shed light on the complex and elegant signaling events triggered by various pathogens.

7.2. Galectins Galectins are β-galactoside-binding lectins, and they are evolutionarily conserved in most organisms (Vasta et al., 2017). Most galectins are transported and secreted to the extracellular space (Dam and Brewer, 2010). Insect galectins are involved in the regulation of immunity against protozoa, bacteria and viruses (Kamhawi et al., 2004; Rao et al., 2016; Sreeramulu et al., 2018). Drosophila contains six candidate galectin genes (Cooper, 2002). Dmgal is the first identified galectin in Drosophila, it contains two carbohydrate recognition domains connected by a peptide linker and it binds to β-galactoside sugars (Pace et al., 2002). Dmgal expression is detected in hemocytes and circulating phagocytic cells, suggesting that Dmgal may participate in microbial recognition and/or phagocytosis

Conflicts of interest The authors declare no conflicts of interest. 6

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Acknowledgements

Filipe, S.R., Tomasz, A., Ligoxygakis, P., 2005. Requirements of peptidoglycan structure that allow detection by the Drosophila Toll pathway. EMBO Rep. 6, 327–333. Franc, N.C., Heitzler, P., Ezekowitz, R.A., White, K., 1999. Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284, 1991–1994. Fullaondo, A., Garcia-Sanchez, S., Sanz-Parra, A., Recio, E., Lee, S.Y., Gubb, D., 2011. Spn1 regulates the GNBP3-dependent Toll signaling pathway in Drosophila melanogaster. Mol. Cell. Biol. 31, 2960–2972. Garver, L.S., Wu, J., Wu, L.P., 2006. The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila. Proc. Natl. Acad. Sci. U. S. A 103, 660. Gendrin, M., Zaidman-Remy, A., Broderick, N.A., Paredes, J., Poidevin, M., Roussel, A., Lemaitre, B., 2013. Functional analysis of PGRP-LA in Drosophila immunity. PLoS One 8, e69742. Gobert, V., Gottar, M., Matskevich, A.A., Rutschmann, S., Royet, J., Belvin, M., Hoffmann, J.A., Ferrandon, D., 2003. Dual activation of the Drosophila toll pathway by two pattern recognition receptors. Science 302, 2126–2130. Gottar, M., Gobert, V., Matskevich, A.A., Reichhart, J.M., Wang, C., Butt, T.M., Belvin, M., Hoffmann, J.A., Ferrandon, D., 2006. Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127, 1425–1437. Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J.A., Ferrandon, D., Royet, J., 2002. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416, 640–644. Guillou, A., Troha, K., Wang, H., Franc, N.C., Buchon, N., 2016. The Drosophila CD36 homologue croquemort is required to maintain immune and gut homeostasis during development and aging. PLoS Pathog. 12, e1005961. Haller, S., Franchet, A., Hakkim, A., Chen, J., Drenkard, E., Yu, S., Schirmeier, S., Li, Z., Martins, N., Ausubel, F.M., Liegeois, S., Ferrandon, D., 2018. Quorum-sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization. EMBO Rep. 19. Hao, Y., Yu, S., Luo, F., Jin, L.H., 2018. Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila. Cell Commun. Signal. : CCS 16 95-95. Hashimoto, Y., Tabuchi, Y., Sakurai, K., Kutsuna, M., Kurokawa, K., Awasaki, T., Sekimizu, K., Nakanishi, Y., Shiratsuchi, A., 2009. Identification of lipoteichoic acid as a ligand for draper in the phagocytosis of Staphylococcus aureus by Drosophila hemocytes. J. Immunol. (Baltimore, Md. : 1950) 183, 7451–7460. Hilu-Dadia, R., Hakim-Mishnaevski, K., Levy-Adam, F., Kurant, E., 2018. Draper-mediated JNK signaling is required for glial phagocytosis of apoptotic neurons during Drosophila metamorphosis. Glia 66, 1520–1532. Hughes, A.L., 2012. Evolution of the betaGRP/GNBP/beta-1,3-glucanase family of insects. Immunogenetics 64, 549–558. Iatsenko, I., Kondo, S., Mengin-Lecreulx, D., Lemaitre, B., 2016. PGRP-SD, an extracellular pattern-recognition receptor, enhances peptidoglycan-mediated activation of the Drosophila imd pathway. Immunity 45, 1013–1023. Igboin, C.O., Tordoff, K.P., Moeschberger, M.L., Griffen, A.L., Leys, E.J., 2011. Porphyromonas gingivalis-host interactions in a Drosophila melanogaster model. Infect. Immun. 79, 449–458. Issa, N., Guillaumot, N., Lauret, E., Matt, N., Schaeffer-Reiss, C., Van Dorsselaer, A., Reichhart, J.-M., Veillard, F., 2018. The circulating protease Persephone is an immune sensor for microbial proteolytic activities upstream of the Drosophila Toll pathway. Mol. Cell 69, 539–550 e536. Kamhawi, S., Ramalho-Ortigao, M., Pham, V.M., Kumar, S., Lawyer, P.G., Turco, S.J., Barillas-Mury, C., Sacks, D.L., Valenzuela, J.G., 2004. A role for insect galectins in parasite survival. Cell 119, 329–341. Kaneko, T., Goldman, W.E., Mellroth, P., Steiner, H., Fukase, K., Kusumoto, S., Harley, W., Fox, A., Golenbock, D., Silverman, N., 2004. Monomeric and polymeric gramnegative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 20, 637–649. Kaneko, T., Yano, T., Aggarwal, K., Lim, J.H., Ueda, K., Oshima, Y., Peach, C., ErturkHasdemir, D., Goldman, W.E., Oh, B.H., Kurata, S., Silverman, N., 2006. PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 7, 715–723. Khush, R.S., Lemaitre, B., 2000. Genes that fight infection: what the Drosophila genome says about animal immunity. Trends Genet. : TIG (Trends Genet.) 16, 442–449. Kim, Y.S., Ryu, J.H., Han, S.J., Choi, K.H., Nam, K.B., Jang, I.H., Lemaitre, B., Brey, P.T., Lee, W.J., 2000. Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells. J. Biol. Chem. 275, 32721–32727. Kirk, P., Bazan, J.F., 2005. Pathogen recognition: TLRs throw us a curve. Immunity 23, 347–350. Kocks, C., Cho, J.H., Nehme, N., Ulvila, J., Pearson, A.M., Meister, M., Strom, C., Conto, S.L., Hetru, C., Stuart, L.M., Stehle, T., Hoffmann, J.A., Reichhart, J.M., Ferrandon, D., Ramet, M., Ezekowitz, R.A., 2005. Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123, 335–346. Kroeger Jr., P.T., Tokusumi, T., Schulz, R.A., 2012. Transcriptional regulation of eater gene expression in Drosophila blood cells. Genesis 50, 41–49 (New York, N.Y. : 2000). Kurant, E., Axelrod, S., Leaman, D., Gaul, U., 2008. Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons. Cell 133, 498–509. Kurata, S., 2014. Peptidoglycan recognition proteins in Drosophila immunity. Dev. Comp. Immunol. 42, 36–41. Kurucz, E., Markus, R., Zsamboki, J., Folkl-Medzihradszky, K., Darula, Z., Vilmos, P., Udvardy, A., Krausz, I., Lukacsovich, T., Gateff, E., Zettervall, C.J., Hultmark, D., Ando, I., 2007. Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes. Curr. Biol. : CB 17, 649–654. Lagueux, M., Perrodou, E., Levashina, E.A., Capovilla, M., Hoffmann, J.A., 2000. Constitutive expression of a complement-like protein in toll and JAK gain-of-function

This work was supported by the National Natural Science Foundation of China (No. 31472019 to X-Q Yu) and Natural Science Foundation of Guangdong Province (No. 2018A030313768 to Y Lu). References Adelman, Z.N., Myles, K.M., 2018. The C-type lectin domain gene family in Aedes aegypti and their role in arbovirus infection. Viruses 10. Agaisse, H., Burrack, L.S., Philips, J.A., Rubin, E.J., Perrimon, N., Higgins, D.E., 2005. Genome-Wide RNAi screen for host factors required for intracellular bacterial infection. Science 309, 1248. Akira, S., Uematsu, S., Takeuchi, O., 2006. Pathogen recognition and innate immunity. Cell 124, 783–801. Ao, J., Ling, E., Yu, X.Q., 2007. Drosophila C-type lectins enhance cellular encapsulation. Mol. Immunol. 44, 2541–2548. Arefin, B., Kucerova, L., Dobes, P., Markus, R., Strnad, H., Wang, Z., Hyrsl, P., Zurovec, M., Theopold, U., 2014. Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins. Journal of innate immunity 6, 192–204. Areschoug, T., Gordon, S., 2009. Scavenger receptors: role in innate immunity and microbial pathogenesis. Cell Microbiol. 11, 1160–1169. Armitage, S.A., Peuss, R., Kurtz, J., 2015. Dscam and pancrustacean immune memory - a review of the evidence. Dev. Comp. Immunol. 48, 315–323. Armitage, S.A., Sun, W., You, X., Kurtz, J., Schmucker, D., Chen, W., 2014. Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure. PLoS One 9, e108660. Armitage, S.A.O., Brites, D., 2016. Chapter 10 - the immune-related roles and the evolutionary history of Dscam in arthropods. In: Malagoli, D. (Ed.), The Evolution of the Immune System. Academic Press, pp. 241–274. Basbous, N., Coste, F., Leone, P., Vincentelli, R., Royet, J., Kellenberger, C., Roussel, A., 2011. The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathway. EMBO Rep. 12, 327–333. Bischoff, V., Vignal, C., Boneca, I.G., Michel, T., Hoffmann, J.A., Royet, J., 2004. Function of the Drosophila pattern-recognition receptor PGRP-SD in the detection of Grampositive bacteria. Nat. Immunol. 5, 1175–1180. Bou Aoun, R., Hetru, C., Troxler, L., Doucet, D., Ferrandon, D., Matt, N., 2011. Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster. Journal of innate immunity 3, 52–64. Bretscher, A.J., Honti, V., Binggeli, O., Burri, O., Poidevin, M., Kurucz, É., Zsámboki, J., Andó, I., Lemaitre, B., 2015. The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster. Biology Open 4, 355–363. Canton, J., Neculai, D., Grinstein, S., 2013. Scavenger receptors in homeostasis and immunity. Nat. Rev. Immunol. 13, 621. Castillo, J.C., Creasy, T., Kumari, P., Shetty, A., Shokal, U., Tallon, L.J., Eleftherianos, I., 2015. Drosophila anti-nematode and antibacterial immune regulators revealed by RNA-Seq. BMC Genomics 16, 519. Chamilos, G., Lewis, R.E., Hu, J., Xiao, L., Zal, T., Gilliet, M., Halder, G., Kontoyiannis, D.P., 2008. Drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis. Proc. Natl. Acad. Sci. U. S. A. 105, 9367–9372. Charroux, B., Capo, F., Kurz, C.L., Peslier, S., Chaduli, D., Viallat-Lieutaud, A., Royet, J., 2018. Cytosolic and secreted peptidoglycan-degrading enzymes in Drosophila respectively control local and systemic immune responses to microbiota. Cell Host Microbe 23, 215–228 e214. Choe, K.M., Werner, T., Stoven, S., Hultmark, D., Anderson, K.V., 2002. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296, 359–362. Chowdhury, M., Li, C.F., He, Z., Lu, Y., Liu, X.S., Wang, Y.F., Ip, Y.T., Strand, M.R., Yu, X.Q., 2019. Toll family members bind multiple Spatzle proteins and activate antimicrobial peptide gene expression in Drosophila. J. Biol. Chem. 294, 10172–10181. Chung, Y.S., Kocks, C., 2011. Recognition of pathogenic microbes by the Drosophila phagocytic pattern recognition receptor Eater. J. Biol. Chem. 286, 26524–26532. Chung, Y.S., Kocks, C., 2012. Phagocytosis of bacterial pathogens. Fly 6, 21–25. Cooper, D.N., 2002. Galectinomics: finding themes in complexity. Biochim. Biophys. Acta 1572, 209–231. Costechareyre, D., Capo, F., Fabre, A., Chaduli, D., Kellenberger, C., Roussel, A., Charroux, B., Royet, J., 2016. Tissue-specific regulation of Drosophila NF-κB pathway activation by peptidoglycan recognition protein SC. Journal of innate immunity 8, 67–80. Cuttell, L., Vaughan, A., Silva, E., Escaron, C.J., Lavine, M., Van Goethem, E., Eid, J.P., Quirin, M., Franc, N.C., 2008. Undertaker, a Drosophila Junctophilin, links Drapermediated phagocytosis and calcium homeostasis. Cell 135, 524–534. Dam, T.K., Brewer, C.F., 2010. Lectins as pattern recognition molecules: the effects of epitope density in innate immunity. Glycobiology 20, 270–279. Dostalova, A., Rommelaere, S., Poidevin, M., Lemaitre, B., 2017. Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps. BMC Biol. 15, 79. El Chamy, L., Leclerc, V., Caldelari, I., Reichhart, J.M., 2008. Sensing of 'danger signals' and pathogen-associated molecular patterns defines binary signaling pathways 'upstream' of Toll. Nat. Immunol. 9, 1165–1170.

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Developmental and Comparative Immunology 102 (2020) 103468

Y. Lu, et al.

Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., Ezekowitz, R.A., 2002. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416, 644–648. Ramet, M., Pearson, A., Manfruelli, P., Li, X., Koziel, H., Gobel, V., Chung, E., Krieger, M., Ezekowitz, R.A., 2001. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15, 1027–1038. Rao, X.J., Wu, P., Shahzad, T., Liu, S., Chen, L., Yang, Y.F., Shi, Q., Yu, X.Q., 2016. Characterization of a dual-CRD galectin in the silkworm Bombyx mori. Dev. Comp. Immunol. 60, 149–159. Royet, J., Gupta, D., Dziarski, R., 2011. Peptidoglycan recognition proteins: modulators of the microbiome and inflammation. Nat. Rev. Immunol. 11, 837–851. Sackton, T.B., Lazzaro, B.P., Clark, A.G., 2010. Genotype and gene expression associations with immune function in Drosophila. PLoS Genet. 6, e1000797. Sackton, T.B., Lazzaro, B.P., Clark, A.G., 2017. Rapid Expansion of Immune-Related Gene Families in the House Fly, Musca domestica. Mol. Biol. Evol. 34, 857–872. Shiratsuchi, A., Mori, T., Sakurai, K., Nagaosa, K., Sekimizu, K., Lee, B.L., Nakanishi, Y., 2012. Independent recognition of Staphylococcus aureus by two receptors for phagocytosis in Drosophila. J. Biol. Chem. 287, 21663–21672. Shokal, U., Eleftherianos, I., 2017a. The Drosophila Thioester containing Protein-4 participates in the induction of the cellular immune response to the pathogen Photorhabdus. Dev. Comp. Immunol. 76, 200–208. Shokal, U., Eleftherianos, I., 2017b. Evolution and Function of Thioester-Containing Proteins and the Complement System in the Innate Immune Response. Front. Immunol. 8, 759. Shokal, U., Eleftherianos, I., 2017c. Thioester-Containing Protein-4 Regulates the Drosophila Immune Signaling and Function against the Pathogen Photorhabdus. Journal of innate immunity 9, 83–93. Shokal, U., Kopydlowski, H., Eleftherianos, I., 2017. The distinct function of Tep2 and Tep6 in the immune defense of Drosophila melanogaster against the pathogen Photorhabdus. Virulence 8, 1668–1682. Shokal, U., Kopydlowski, H., Harsh, S., Eleftherianos, I., 2018. Thioester-Containing Proteins 2 and 4 Affect the Metabolic Activity and Inflammation Response in Drosophila. Infect. Immun. 86. Smith, P.H., 2012. Dscam Gene Expression in Invertebrate Immunity : Alternative Splicing in Response to Diverse Pathogens. University of Edinburgh, The University of Edinburgh. Somogyi, K., Sipos, B., Penzes, Z., Kurucz, E., Zsamboki, J., Hultmark, D., Ando, I., 2008. Evolution of genes and repeats in the Nimrod superfamily. Mol. Biol. Evol. 25, 2337–2347. Sreeramulu, B., Arumugam, G., Paulchamy, R., Karuppiah, H., Sundaram, J., 2018. betaGalactoside binding lectin from caddisfly larvae, Stenopsyche kodaikanalensis with selective modes of antibacterial activity: Purification and characterization. Int. J. Biol. Macromol. 115, 1033–1045. Strand, M.R., 2008. The insect cellular immune response. Insect Sci. 15, 1–14. Stroschein-Stevenson, S.L., Foley, E., O'Farrell, P.H., Johnson, A.D., 2006. Identification of Drosophila gene products required for phagocytosis of Candida albicans. PLoS Biol. 4, e4. Stuart, L.M., Deng, J., Silver, J.M., Takahashi, K., Tseng, A.A., Hennessy, E.J., Ezekowitz, R.A., Moore, K.J., 2005. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170, 477–485. Sun, J.J., Lan, J.F., Zhao, X.F., Vasta, G.R., Wang, J.X., 2017. Binding of a C-type lectin's coiled-coil domain to the Domeless receptor directly activates the JAK/STAT pathway in the shrimp immune response to bacterial infection. PLoS Pathog. 13, e1006626. Takehana, A., Katsuyama, T., Yano, T., Oshima, Y., Takada, H., Aigaki, T., Kurata, S., 2002. Overexpression of a pattern-recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in Drosophila larvae. Proc. Natl. Acad. Sci. U. S. A. 99, 13705–13710. Takehana, A., Yano, T., Mita, S., Kotani, A., Oshima, Y., Kurata, S., 2004. Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J. 23, 4690–4700. Tanji, T., Ohashi-Kobayashi, A., Natori, S., 2006. Participation of a galactose-specific Ctype lectin in Drosophila immunity. Biochem. J. 396, 127–138. Tavignot, R., Chaduli, D., Djitte, F., Charroux, B., Royet, J., 2017. Inhibition of a NFkappaB/Diap1 pathway by PGRP-LF is required for proper apoptosis during Drosophila development. PLoS Genet. 13, e1006569. Vasta, G.R., Feng, C., Gonzalez-Montalban, N., Mancini, J., Yang, L., Abernathy, K., Frost, G., Palm, C., 2017. Functions of galectins as 'self/non-self'-recognition and effector factors. Pathogens and disease 75. Vlisidou, I., Dowling, A.J., Evans, I.R., Waterfield, N., ffrench-Constant, R.H., Wood, W., 2009. Drosophila embryos as model systems for monitoring bacterial infection in real time. PLoS Pathog. 5, e1000518. Wang, J., Song, X., Wang, M., 2018a. Peptidoglycan recognition proteins in hematophagous arthropods. Dev. Comp. Immunol. 83, 89–95. Wang, L., Gilbert, R.J., Atilano, M.L., Filipe, S.R., Gay, N.J., Ligoxygakis, P., 2008. Peptidoglycan recognition protein-SD provides versatility of receptor formation in Drosophila immunity. Proc. Natl. Acad. Sci. U. S. A. 105, 11881–11886. Wang, L., Weber, A.N., Atilano, M.L., Filipe, S.R., Gay, N.J., Ligoxygakis, P., 2006. Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA. EMBO J. 25, 5005–5014. Wang, Q., Ren, M., Liu, X., Xia, H., Chen, K., 2019. Peptidoglycan recognition proteins in insect immunity. Mol. Immunol. 106, 69–76. Wang, W., Song, X., Wang, L., Song, L., 2018b. Pathogen-derived carbohydrate recognition in Molluscs immune defense. Int. J. Mol. Sci. 19. Watson, F.L., Puttmann-Holgado, R., Thomas, F., Lamar, D.L., Hughes, M., Kondo, M.,

mutants of Drosophila. Proc. Natl. Acad. Sci. U. S. A. 97, 11427–11432. Lamiable, O., Arnold, J., de Faria, I., Olmo, R.P., Bergami, F., Meignin, C., Hoffmann, J.A., Marques, J.T., Imler, J.L., 2016. Analysis of the contribution of hemocytes and autophagy to Drosophila antiviral immunity. J. Virol. 90, 5415–5426. Lazzaro, B.P., 2005. Elevated polymorphism and divergence in the class C scavenger receptors of Drosophila melanogaster and D. simulans. Genetics 169, 2023–2034. Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25, 697–743. Leone, P., Bischoff, V., Kellenberger, C., Hetru, C., Royet, J., Roussel, A., 2008. Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan. Mol. Immunol. 45, 2521–2530. Leulier, F., Parquet, C., Pili-Floury, S., Ryu, J.H., Caroff, M., Lee, W.J., Mengin-Lecreulx, D., Lemaitre, B., 2003. The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat. Immunol. 4, 478–484. Low, L.Y., Yang, C., Perego, M., Osterman, A., Liddington, R., 2011. Role of net charge on catalytic domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of phage lysins. J. Biol. Chem. 286, 34391–34403. Lu, A., Zhang, Q., Zhang, J., Yang, B., Wu, K., Xie, W., Luan, Y.X., Ling, E., 2014. Insect prophenoloxidase: the view beyond immunity. Front. Physiol. 5, 252. MacLeod, D.T., Nakatsuji, T., Wang, Z., di Nardo, A., Gallo, R.L., 2015. Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes. J. Investig. Dermatol. 135, 142–150. Maillet, F., Bischoff, V., Vignal, C., Hoffmann, J., Royet, J., 2008. The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation. Cell Host Microbe 3, 293–303. Matskevich, A.A., Quintin, J., Ferrandon, D., 2010. The Drosophila PRR GNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll-pathway activation function. Eur. J. Immunol. 40, 1244–1254. Melcarne, C., Ramond, E., Dudzic, J., J Bretscher, A., Kurucz, E., Andó, I., Lemaitre, B., 2018. Two Nimrod Receptors, NimC1 and Eater, Synergistically Contribute to Bacterial Phagocytosis in Drosophila melanogaster. Mellroth, P., Karlsson, J., Steiner, H., 2003. A scavenger function for a Drosophila peptidoglycan recognition protein. J. Biol. Chem. 278, 7059–7064. Mellroth, P., Steiner, H., 2006. PGRP-SB1: an N-acetylmuramoyl L-alanine amidase with antibacterial activity. Biochem. Biophys. Res. Commun. 350, 994–999. Michel, T., Reichhart, J.M., Hoffmann, J.A., Royet, J., 2001. Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414, 756–759. Mishima, Y., Quintin, J., Aimanianda, V., Kellenberger, C., Coste, F., Clavaud, C., Hetru, C., Hoffmann, J.A., Latge, J.P., Ferrandon, D., Roussel, A., 2009. The N-terminal domain of Drosophila Gram-negative binding protein 3 (GNBP3) defines a novel family of fungal pattern recognition receptors. J. Biol. Chem. 284, 28687–28697. Nakamoto, M., Moy, R.H., Xu, J., Bambina, S., Yasunaga, A., Shelly, S.S., Gold, B., Cherry, S., 2012. Virus recognition by Toll-7 activates antiviral autophagy in Drosophila. Immunity 36, 658–667. Nehme, N.T., Quintin, J., Cho, J.H., Lee, J., Lafarge, M.C., Kocks, C., Ferrandon, D., 2011. Relative roles of the cellular and humoral responses in the Drosophila host defense against three gram-positive bacterial infections. PLoS One 6, e14743. Neyen, C., Runchel, C., Schupfer, F., Meier, P., Lemaitre, B., 2016. The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors. Nat. Immunol. 17, 1150–1158. Nilsson, C.L., 2011. Lectins: Analytical Technologies. Elsevier Science. Nonaka, S., Nagaosa, K., Mori, T., Shiratsuchi, A., Nakanishi, Y., 2013. Integrin alphaPS3/ betanu-mediated phagocytosis of apoptotic cells and bacteria in Drosophila. J. Biol. Chem. 288, 10374–10380. Okuda, K., Tong, M., Dempsey, B., Moore, K.J., Gazzinelli, R.T., Silverman, N., 2016. Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation. PLoS Pathog. 12, e1005669. Pace, K.E., Lebestky, T., Hummel, T., Arnoux, P., Kwan, K., Baum, L.G., 2002. Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues. J. Biol. Chem. 277, 13091–13098. Paredes, J.C., Welchman, D.P., Poidevin, M., Lemaitre, B., 2011. Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 35, 770–779. Pearson, A., Lux, A., Krieger, M., 1995. Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 92, 4056–4060. Pees, B., Yang, W., Zarate-Potes, A., Schulenburg, H., Dierking, K., 2016. High Innate Immune Specificity through Diversified C-Type Lectin-Like Domain Proteins in Invertebrates. Journal of innate immunity 8, 129–142. Peuss, R., Wensing, K.U., Woestmann, L., Eggert, H., Milutinovic, B., Sroka, M.G., Scharsack, J.P., Kurtz, J., Armitage, S.A., 2016. Down syndrome cell adhesion molecule 1: testing for a role in insect immunity, behaviour and reproduction. Royal Society open science 3, 160138. Philips, J.A., Rubin, E.J., Perrimon, N., 2005. Drosophila RNAi Screen Reveals CD36 Family Member Required for Mycobacterial Infection. Science 309, 1251. Pili-Floury, S., Leulier, F., Takahashi, K., Saigo, K., Samain, E., Ueda, R., Lemaitre, B., 2004. In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults. J. Biol. Chem. 279, 12848–12853. PrabhuDas, M.R., Baldwin, C.L., Bollyky, P.L., Bowdish, D.M.E., Drickamer, K., Febbraio, M., Herz, J., Kobzik, L., Krieger, M., Loike, J., McVicker, B., Means, T.K., Moestrup, S.K., Post, S.R., Sawamura, T., Silverstein, S., Speth, R.C., Telfer, J.C., Thiele, G.M., Wang, X.-Y., Wright, S.D., El Khoury, J., 2017. A Consensus Definitive Classification of Scavenger Receptors and Their Roles in Health and Disease. J. Immunol. 198, 3775.

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Developmental and Comparative Immunology 102 (2020) 103468

Y. Lu, et al.

W.E., Fukase, K., Silverman, N., Yoshimori, T., Kurata, S., 2008. Autophagic control of listeria through intracellular innate immune recognition in Drosophila. Nat. Immunol. 9, 908–916. Yi, H.Y., Chowdhury, M., Huang, Y.D., Yu, X.Q., 2014. Insect antimicrobial peptides and their applications. Appl. Microbiol. Biotechnol. 98, 5807–5822. Zaidman-Remy, A., Herve, M., Poidevin, M., Pili-Floury, S., Kim, M.S., Blanot, D., Oh, B.H., Ueda, R., Mengin-Lecreulx, D., Lemaitre, B., 2006. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24, 463–473. Zsámboki, J., Gábor, C., Honti, V., Pintér, L., Bajusz, I., Galgoczy, L., Andó, I., Kurucz, E., 2013. Drosophila Nimrod Proteins Bind Bacteria.

Rebel, V.I., Schmucker, D., 2005. Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309, 1874–1878. Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H., Hultmark, D., 2000. A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 97, 13772–13777. Wojtowicz, W.M., Wu, W., Andre, I., Qian, B., Baker, D., Zipursky, S.L., 2007. A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains. Cell 130, 1134–1145. Xia, X., You, M., Rao, X.J., Yu, X.Q., 2018. Insect C-type lectins in innate immunity. Dev. Comp. Immunol. 83, 70–79. Yano, T., Mita, S., Ohmori, H., Oshima, Y., Fujimoto, Y., Ueda, R., Takada, H., Goldman,

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