In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Sp?tzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.     

Requirements of peptidoglycan structure that allow detection by the Drosophila Toll pathway

The Drosophila immune system is able to discriminate between classes of bacteria. Detection of Gram-positive bacteria involves a complex of two pattern recognition receptors: peptidoglycan recognition protein SA (PGRP-SA) and Gram-negative binding protein 1 (GNBP1). These activate the Toll signalling pathway. To define the cell wall components sensed by the host, we used highly purified peptidoglycan fragments of two principal Gram-positive bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae. We report that in both peptidoglycans, the minimal structure needed to activate the Toll pathway is a muropeptide dimer and that the free reducing end of the N-acetyl muramic acid residues of the muropeptides is essential for activity. Monomeric muropeptides were inactive and inhibitory in combination with dimers. Finally, peptidoglycan was degraded by the haemolymph of wild-type but not GNBP1 mutant flies. We suggest a model whereby GNBP1 is involved in the hydrolysis of Gram-positive peptidoglycan producing new glycan reducing ends, which are subsequently detected by PGRP-SA.     

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.     

Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila

In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.     

Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation

Unlike mammalian Toll-like Receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi . In the present study, we have used an in vivo RNAi strategy to inactivate 75 distinct Drosophila SP genes. We then screened this collection for SPs regulating the activation of the Toll pathway by gram-positive bacteria. Here, we report the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs.     

Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.     

Positive and negative regulation of the Drosophila immune response

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.     

The N-terminal Domain of Drosophila Gram-negative Binding Protein 3 (GNBP3) Defines a Novel Family of Fungal Pattern Recognition Receptors

Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. Here, we report that GNBP3 N-terminal domain binds to fungi upon identifying long chains of β-1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for β-glucan recognition.The activation of the immune response is energetically costly and may be detrimental to the host, especially when inappropriately triggered. Therefore, the reliable detection of infections is a step of paramount importance in the immune response. To achieve the task of detecting potentially hazardous microorganisms, the innate immune system relies on several strategies. One of them is to sense both pathogenic and nonpathogenic microorganisms thanks to pattern recognition receptors (PRRs)⁴ that recognize intrinsic microbial molecular “signatures” (1). These immune receptors have been selected during evolution for their ability to bind to essential, conserved, structural components of the microorganisms such as flagellins, peptidoglycans of bacteria, lipopolysaccharides of Gram-negative bacteria, lipoteichoic acids of Gram-positive bacteria, and β-glucans of the fungal cell wall (2, 3). Examples of mammalian PRRs include Toll-like receptors (4), intracellular receptors of the NOD family (5), peptidoglycan recognition proteins (PGRPs) (6), and the membrane-bound Dectin-1 receptor, which detects fungal β-glucans (7).One important arm of the innate immunity in Drosophila is a potent systemic response that relies on the synthesis in the fat body (a functional equivalent of the mammalian liver) of potent antimicrobial peptides (AMPs) that are secreted in the hemolymph where they attack invading microorganisms. Genetic analysis has delineated two major regulatory pathways of NF-κB type that control the expression of AMP genes (8). The immune deficiency (imd) pathway is mostly required in the host defense against Gram-negative bacteria (9) and is triggered by PRRs of the PGRP family, namely PGRP-LC (10) and PGRP-LE (11). The Toll pathway is essential for fighting fungal and some Gram-positive bacterial infections (12, 13). Toll, the funding member of the Toll-like receptor family, is not itself a PRR. Rather, it is activated by a ligand of the nerve growth factor family, the Spätzle cytokine. To bind to the Toll receptor, Pro-Spätzle needs to be proteolytically processed by a protease, the Spätzle-processing enzyme (SPE) (14), which is itself activated by upstream proteolytic cascades. One such cascade is activated in response to a Gram-positive bacterial challenge by a complex of PGRP-SA, PGRP-SD, and Gram-negative binding protein 1 (GNBP1) (13, 15, 16). Flies deficient for either PGRP-SA or GNBP1 are deficient in Toll pathway activation and are susceptible to infections by several Gram-positive bacterial species but not to fungal infections. In contrast, flies mutant for GNBP3, another gene encoding a GNBP family member, fail to activate the Toll pathway in response to killed fungi and succumb rapidly to fungal but not bacterial infections (17). GNBP3 is thought to activate a proteolytic cascade, which partially overlaps that triggered by the GNBP1·PGRP-SA complex (18). Even though they belong to the same family and activate the same pathway, GNBP1 and GNBP3 are required for sensing distinct classes of microorganisms.The founding member of the GNBP family, a 50-kDa protein found in hemolymph of Bombyx mori and originally named p50, was characterized as a gram-negative (Escherichia coli) binding protein (19); hence, its name. However, it has become clear that GNBPs belong to the family of β-glucan recognition proteins (βGRP) that had first been purified on their ability to trigger the prophenol oxidase cascade (a wound response that leads to melanization at the injury site) in response to fungal infections (20). Members of the GNBP/βGRP family are extracellular proteins composed of a small N-terminal domain of about 100 residues and a longer C-terminal domain of about 350 residues (21, 22). In the insect Plodia interpunctella, both domains of βGRP bind to laminarin, a soluble β-1,3-glucan with a high affinity (K_A in the 10⁸ m⁻¹ range) (23) which is in the same range as that of the Factor G of the Japanese horseshoe crab (24). The latter factor is used as a diagnostic reagent for the detection of glucans. The C-terminal domain displays sequence similarity to bacterial glucanases, yet the catalytic residues have not been conserved, suggesting that this domain has been selected during evolution for its ability to bind to glucans (21, 22). The N-terminal domain defines a novel β-1,3-glucan binding domain that binds to curdlan, an insoluble linear β-1,3-glucan polymer, a property that the C-terminal glucanase-like domain lacks (21). Full-length recombinant GNBP/βGRPs have been reported to bind to bacteria, lipopolysaccharides, or lipoteichoic acids (19, 22, 23, 25). Although the domain(s) that mediates these interactions has not been thoroughly mapped, it appears that the N-terminal P. interpunctella β-1,3-glucan domain is not required for binding to these bacterial compounds (23).Numerous three-dimensional structures of PGRPs, in some cases complexed with their ligands, have been reported (26–29). In contrast, this knowledge is currently lacking as regarding GNBPs. As a first step toward elucidating the structure/function relationships of GNBPs, we report here that a recombinant polypeptide encoding the N-terminal domain of GNBP3 binds to fungi and to long β-1,3-glucan chains but not to short laminarioligosaccharides. The determination of the crystal structure of GNBP3 N-terminal domain reveals an immunoglobulin fold in which the β-glucan binding site is masked by a lid, which is likely to be displaced by long polysaccharide chains.     

Tissue and stage-specific expression of the Tolls in Drosophila embryos

The Drosophila transmembrane receptor Toll plays a key role in specifying the dorsoventral axis of the embryo. At later stages of development, it controls the immune response of the fly to fungal and Gram-positive bacterial infections. The Drosophila genome has a total of nine Toll-like genes, including the previously characterized Toll (Toll-1) and 18-wheeler (Toll-2). Here we describe the embryonic expression patterns of the seven Toll-like genes Toll-3 through Toll-9. We find that these genes have distinct expression domains and that their expression is dynamically changing throughout embryonic development. This complex and tissue-specific regulation of Toll-like gene expression strongly suggests a role in embryonic development for most Drosophila Tolls. The evolving picture on the Toll family members in Drosophila contrasts with that of mammalian Toll-like receptors, which are predominantly expressed in immune responsive cells where their activation occurs via microbial structural determinants.     

Using non-mammalian hosts to study fungal virulence and host defense

Non-mammalian hosts have been used to study host-fungal interactions. Hosts such as Drosophila melanogaster, Caenorhabditis elegans, Acathamoeba castellanii, Dictyostelium discoideum, and Galleria mellonella have provided means to examine the physical barriers, cellular mechanisms and molecular elements of the host response. The Drosophila host-response to fungi is mediated through the Toll pathway, whereas in C. elegans the host-response is TIR-1-dependent. Virulence traits that are involved in mammalian infection are important for the interaction of fungi with these hosts. Screening of fungal virulence traits using mutagenized fungi to determine changes in fungal infectivity of non-mammalian hosts has been used to identify novel virulence proteins used to infect C. elegans such as Kin1 (a serine/threonine protein kinase) and Rom2 (a Rho1 guanyl-nucleotide exchange factor) from Cryptococcus neoformans. These heterologous non-mammalian hosts highlight the similarities and differences between different hosts in fungal pathogenesis and they complement studies in mammalian systems and those using other genetic approaches.     

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.     

Toll-like receptors: lessons from knockout mice

The Toll signalling pathway, which is required for establishment of dorsoventral polarity in Drosophila embryos, plays an important role in the response to microbial infections. Recently, Toll-like receptors (TLRs) have also been identified in mammals. TLR4 has been shown to function as the transmembrane component of the lipopolysaccharide receptor, while TLR2 recognizes peptidoglycans from Gram-positive bacteria, lipoproteins and yeast. Although various microbial cell-wall components are recognized by different receptors, all of these responses are abrogated in MyD88-deficient cells. These results show that different TLRs recognize different microbial cell-wall components, and that MyD88 is an essential signalling molecule shared among interleukin-1 receptor/Toll family members.     

Toll-like receptors and innate antifungal responses

The mammalian Toll-like receptors (TLRs) are homologues of Drosophila Toll and constitute a novel protein family involved in the mediation of innate immunity and the activation of adaptive immunity. Analysis of infection with human pathogenic fungi Candida albicans and Aspergillus fumigatus implicated TLR2 and TLR4 in elicitation of immune responses. Cryptococcus neoformans is recognized by a process that uses TLR4. C. albicans induces immunostimulation through causative agents, such as mannan or its structural derivatives (e.g. phospholipomannan), which are recognized by the immune system as pathogen-associated molecular patterns and are located in the cell wall of fungi. Secreted aspartic proteinases represent a key virulence factor that contributes to the ability of C. albicans to cause mucosal and disseminated infections, and might be a further potential stimulator of TLRs. Simultaneous activation of other pattern recognition receptors collaborating with TLRs illustrates the cooperation of various chains within ligand-specific receptor complexes for the recognition of fungal pathogens and their cell wall components.     

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.     

家蝇微生物感染对RNA干扰GNBP3基因的作用

为了研究家蝇Musca domestica微生物感染对RNA干扰GNBP3基因的作用,评价微生物感染后对下游抗菌肽水平的影响,本研究用RNA干扰技术沉默家蝇GNBP3基因探索最佳沉默时间及效果,检测RNA干扰后家蝇幼虫存活及化蛹情况,通过微生物喂食途径感染,采用实时荧光定量PCR方法检测抗菌肽基因(cecropins、...     

Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we have used this method to silence components of a major signaling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bacterial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.