InteractiveFly: GeneBrief

eater and Nimrod C1: Biological Overview | References

Hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. Located on the surface of circulating and sessile plasmatocytes, Nimrod C1 (NimC1) is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibit the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater, which is also located on phagocytic cells and mediates resistence to bacteria (Kocks, 2005), Draper from Drosophila, and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome (Kurucz, 2007).

A set of monoclonal antibodies has been generated that define hemocyte subsets and identify hemocyte-specific molecules (Kurucz, 2003; Vilmos, 2004), putative regulators of hemocyte development and function. This study used antibodies for a plasmatocyte-specific antigen, P1, to identify a novel transmembrane protein. P1 is defined by two monoclonal antibodies, P1a (Vilmos, 2004) and P1b, which recognize two different epitopes on the same antigen. This antigen is present in a subpopulation of circulating hemocytes in the l(3)mbn-1 hemocyte-overproducing mutant (Konrad, 1994). Indirect immunofluorescence analysis of live larval hemocytes shows that both epitopes are expressed on the cell surface on the majority of larval hemocytes, all with plasmatocyte morphology, and are absent on the lamellocytes and the crystal cells, the two other major classes of larval hemocytes. FACS analysis showed that P1 is present on approximately 80% of the circulating hemocytes in l(3)mbn-1 larvae, corresponding to the plasmatocyte fraction in this mutant, which spontaneously produces large numbers of all three classes of hemocytes. It is found on approximately 90% of the hemocytes in first instar Oregon-R larvae immediately after hatching, 94% in second instar, and 97% in late third instar larvae. It is also expressed in the adult, although it is essentially absent from the sessile population at that stage. P1 was not detected in embryonic hemocytes. The P1-positive cells phagocytose bacteria and produce antimicrobial peptides, suggesting that they are involved in the antimicrobial defense. Cells with lamellocyte morphology, lacking P1, are phagocytically inactive (Kurucz, 2007).

Most larval hemocytes are believed to originate directly from a population of embryonic hemocytes, although evidence has been presented that a second population of hemocytes develops in a specialized hematopoietic tissue, the lymph glands. In late third instar larvae, P1 was found expressed in islands of cells in the anterior lobes of the lymph glands as well as in islands of sessile subepidermal hemocytes. Because actively dividing P1-positive cells were also found among the circulating hemocytes, it is concluded that plasmatocytes, defined as P1-positive cells, are produced in both populations of larval hemocytes (Kurucz, 2007).

A mixture of the two P1-specific antibodies was used to isolate the P1 antigen by immunoprecipitation. A 90-100 kDa silver-stained protein band, corresponding to the P1 antigen, was excised, digested with trypsin, and analyzed with MALDI-TOF mass spectrometry. A peptide with MH⁺ at m/z 912.54 identified a sequence, VIPYQHR from a predicted Drosophila gene, CG8942, hereafter called nimrod C1 (nimC1). A single sequenced cDNA clone, LP05465 (accession AY119029), defines a 2040 nucleotide nimC1 transcript. It encodes a 620 amino acid open reading frame, NimC1, with an N-terminal signal peptide and then a putative single-pass transmembrane protein of 65 kDa. The larger size observed on the western blot indicates that NimC1 may be glycosylated (Kurucz, 2007).

To verify that the nimC1 gene encodes P1, it was expressed in the P1-negative Drosophila cell line, Schneider-2 (S2), under the control of an inducible promoter. The NimC1 protein could be detected on the plasma membrane of live transfected S2 cells with the antibodies P1a and P1b. The overexpressed protein is slightly smaller than the antigen found in l(3)mbn-1 hemocytes, presumably because of differences in glycosylation. No signal was detected in cells transfected with the empty vector. Overexpression of NimC1 made the S2 cells highly adherent. They formed aggregates, and their growth was retarded (Kurucz, 2007).

The identity of the P1 antigen was further verified by silencing the nimC1 gene product by RNAi. A UAS-nimC1-IR hairpin construct was expressed in transgenic larvae by using the hemocyte-specific Hemese-Gal4 (He-Gal4) driver (Zettervall, 2004). This resulted in a significant decrease of the P1 expression on the plasmatocytes. Two other independent transgenic lines gave similar results. However, P1 is still expressed in a minor population of cells, probably corresponding to the approximately 20% of the hemocytes that do not express the He-Gal4 driver. FACS analysis confirms that P1 expression on the cell surface is correspondingly reduced in the He-GAL4xUAS-nimC1-IR cross compared to the parental He-GAL4 or UAS-nimC1-IR stocks. Hemese antigen expression was normal. The results of these loss-of-function and gain-of-function experiments clearly show that the P1 antigen is a product of the nimC1 gene (Kurucz, 2007).

Because NimC1 is expressed exclusively in phagocytic cells, its possible role in phagocytosis was tested. Initial attempts to block phagocytosis in Oregon-R hemocytes with P1 antibodies were negative, but when NimC1 expression was suppressed with the hairpin construct, a dramatic effect was seen on the phagocytic capacity of the plasmatocytes. The phagocytic index for Staphylococcus aureus bacteria decreased to approximately one-third of the controls. However, the phagocytosis of Escherichia coli was not significantly affected in these experiments. NimC1 suppression had no effect on the gross binding of bacteria at 4°C, showing that NimC1 is involved in phagocytosis but contributes little to the overall adhesion of bacteria to the cells. Further evidence for the role of NimC1 in phagocytosis was obtained by CuSO₄-induced overexpression of NimC1 in Schneider-2 cells, a cell line that does not express NimC1. This stimulated the uptake of both S. aureus and E. coli. The mean fluorescence intensity of the whole (M3 gated) population increased by 1.91-fold for E. coli and by 2.45-fold for S. aureus. It is concluded that NimC1 is a major factor in the phagocytosis of S. aureus by plasmatocytes and that it may also play a redundant role in the phagocytosis of E. coli. The fact that NimC1 is not expressed in S2 cells may explain why it was not detected in previous screens for phagocytosis receptors (Kurucz, 2007).

NimC1 overexpression did not significantly affect the total binding of bacteria to the cell surface. The background of nonspecific adhesion of bacteria to the cell surface may have obscured the binding to NimC1. Alternatively, it is possible that NimC1 may act indirectly, as a coreceptor or at a later stage in the phagocytic process (Kurucz, 2007).

The extracellular region of the NimC1 protein has ten repeats of an EGF-like motif with six cystein residues; the motif is followed by a nonrepetitive cystein-rich domain, a predicted transmembrane domain, and a short intracellular domain. The EGF-like repeats, here called NIM repeats, have a well-conserved consensus sequence CxPxCxxxCxNGxCxxPxxCxCxxGY and are separated by variable loops of typically. This motif differs significantly from the typical EGF repeat, xxxxCx_2-7Cx_1-4(G/A)xCx_1-13ttaxCxCxxGax_1-6GxxCx, and is shifted by one cystein unit compared to the latter (Kurucz, 2007).

The nimC1 gene is located at 34E5 on chromosome 2, immediately 5' of the hemocyte-specific Hemese gene, and within a cluster of ten genes that all have NIM repeats. These nimrod genes also share with nimC1 a short conserved motif, CCxGY, immediately preceding the first NIM repeat. Similar sequences are also present in the Drosophila genes draper at 62B, CG7447 at 64B, and eater at 97E (Kurucz, 2007).

The ten nimrod genes encode three different classes of proteins. The nimA gene encodes a protein with an EMI domain, a possible protein-protein interaction module that was first named after its presence in proteins of the EMILIN family (Callebaut, 2003). The EMI domain is followed by a single NIM repeat, two copies of another atypical EGF-like repeat with eight cysteins, a putative transmembrane region, and a relatively large intracellular domain. A similar arrangement is found in the products of the ced-1 gene in C. elegans and draper in Drosophila. By contrast, the nimB1-nimB5 gene products lack membrane anchors and are probably exported. They have one to eight NIM repeats and share a weakly conserved sequence at the amino terminal, but they have no other known motifs. Finally, nimC1-nimC4 gene products represent a third class. They are transmembrane proteins, with the possible exception of nimC3 for which a 3' exon has not yet been identified. They have 2-16 NIM repeats and also show additional sequence conservation at the amino terminal. RT-PCR assays indicate that all nimrod genes except nimA are transcribed in hemocytes, both in wild-type larvae and in the hemocyte-overproducing l(3)mbn-1 mutant (Kurucz, 2007).

Homologous nimrod gene clusters with similar genomic organization can also be identified in the sequenced genomes of the mosquito, Anopheles gambiae and the honeybee, Apis mellifera. However, the number of nimrod homologs is smaller in Anopheles and Apis: one nimA-like, one nimB-like, and two nimC-like genes, but no Hemese homolog. The class-specific N-terminal motifs are also present, except in the Anopheles nimC-like genes. Homologs of draper and CG7447 can also be found in Anopheles and Apis, and there is a possible eater homolog in the mosquito but not in the honeybee (Kurucz, 2007).

A database search shows that proteins with a CCxGY motif, followed by one or several NIM repeats, can be found in many organisms, including man. Their functions are known in a few cases, all of them related to phagocytosis, microbial binding, or both. Recently, Kocks (2005) showed that the Drosophila protein Eater is directly involved in the phagocytosis of bacteria. Eater is very similar to the NimC class of proteins, with a CCxGY motif, 28-32 NIM repeats, and a transmembrane region, but it lacks the conserved region at the amino terminus. Furthermore, the NimA-like proteins CED-1 in C. elegans and Draper in D. melanogaster are both receptors for phagocytosis of apoptotic cells. A series of ced-1-like genes in mammals, such as MEGF10 in humans and Jedi in mouse, may have the same function. Close relatives of the nimB genes have been described from the silkmoth, Bombyx mori, and from a beetle, Holotrichia diomphalia. The latter encodes an LPS-binding protein that agglutinates bacteria in the hemolymph (Ju, 2006). Furthermore, a likely ortholog to the nimC2 gene in flesh fly, Sarcophaga peregrina, encodes a 120 kDa protein that was proposed to act as a scavenger receptor in hemocytes of this fly (Nishikawa, 2001). Thus, Nimrod C1 belongs to a diverse class of proteins, many of which are phagocytosis receptors or bacteria-binding factors (Kurucz, 2007).

Nimrod C1 and the related Eater protein represent a novel class of putative phagocytosis receptors, characterized by a unique variant of the EGF repeat: the NIM repeat. It remains to be seen whether NimC1, like Eater, binds directly to bacteria, but these experiments show that it is a major factor in the phagocytosis of S. aureus and that it can also contribute to the phagocytosis of E. coli. Two other potential receptors for phagocytosis of bacteria have previously been described from Drosophila, and they are Peptidoglycan recognition protein LC and Scavenger receptor class C, type I. There is probably much redundancy among the factors involved in the phagocytosis of bacteria. Proteins with EGF-like repeats play an important role in this process, in insects and perhaps in man (Kurucz, 2007).

Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila

Phagocytosis is a complex, evolutionarily conserved process that plays a central role in host defense against infection. A predicted transmembrane protein, Eater, has been identified that is involved in phagocytosis in Drosophila. Transcriptional silencing of the eater gene in a macrophage cell line led to a significant reduction in the binding and internalization of bacteria. Moreover, the N terminus of the Eater protein mediates direct microbial binding which can be inhibited with scavenger receptor ligands (acetylated and oxidized low-density lipoproteins). In vivo, eater expression is restricted to blood cells. Flies lacking the eater gene displayed normal responses in NF-kappaB-like Toll and IMD signaling pathways but show impaired phagocytosis and decreased survival after bacterial infection. These results suggest that Eater is a major phagocytic receptor for a broad range of bacterial pathogens in Drosophila and provide a powerful model to address the role of phagocytosis in vivo (Kocks, 2005; full text of article).

Mammalian phagocyte-microbe interactions depend upon recognition of microbe surfaces by a variety of different phagocytic receptors. Among these are the more specialized opsonin-dependent Fc and complement receptors but also opsonin-independent receptors such as the mannose receptor and a group of structurally unrelated receptors termed scavenger receptors. The latter are defined by their ability to recognize a variety of charged, polyanionic ligands (including modified LDLs) and may constitute the most primitive type of microbial phagocytic receptors. Commensurate with the latter idea, one would predict that animals which lack adaptive immunity have a more limited repertoire of specialized phagocytic receptors and thus might rely on scavenger-like receptors to recognize a broad range of microbes. Eater is a predicted transmembrane protein that appears to be a predominant molecule involved in Drosophila macrophage-mediated phagocytosis (Kocks, 2005).

It is suggested that Eater functions as a cell surface bound pattern recognition receptor in the initial steps of phagocytosis -- the recognition and binding of pathogens. Consistent with its predicted structure as a type I membrane protein, a functional signal sequence and functional N-glycosylation sites, loss of Eater expression in Drosophila macrophages resulted in a strong impairment of bacterial binding to the cell surface. The extracellular part of Eater consists mostly of EGF-like repeats, among which the four most N-terminal repeats show high variability in their surface loops and are preceded by a 40 amino acid domain. This region of Eater binds bacteria, supporting the idea that Eater functions as a receptor that directly interacts with microbes and that the N-terminal 199 amino acids of Eater participate in ligand binding. It remains to be determined whether in Eater EGF-like repeats serve a predominantly structural function such as in scavenger receptors of the LDL receptor (CD91/LRP) family or in integrins, where they help to expose the ligand binding site, or whether they directly participate in microbial recognition (Kocks, 2005).

Eater seems capable of recognizing multiple ligands, a binding behavior that is reminiscent of scavenger receptors and also of lipopolysaccharide binding protein (LBP), an acute phase protein structurally unrelated to Eater. Binding of ligands by LBP is directed toward recognition of lipids linked to carbohydrate structures and may be explained by a propensity to bind iterative polyanionic groups. This concept is consistent with the finding that typical scavenger receptor ligands such as modified low-density lipoproteins inhibit N-terminal Eater binding. Moreover, preliminary results suggest that Eater is involved in lipopolysaccharide recognition. Thus, Eater may add another structural variation to the theme of multiligand recognition, and further analysis of Eater may provide insights into the structural basis of multiligand binding in general (Kocks, 2005).

Mammalian class A scavenger receptors possess only a short intracytoplasmic tail and are thought to contribute mainly to microbial binding, while coreceptors generate the signals required for particle internalization. The underlying signaling pathways are poorly understood. Similar to these receptors, Eater possesses only a short intracellular tail, and it needs to be addressed whether Eater alone is sufficient for particle recognition and internalization. In any event, the study of downstream effectors for Eater should lead to insights into evolutionarily conserved internalization pathways of phagocytosis (Kocks, 2005).

Homology searches at the amino acid level failed to pinpoint a clear mammalian homolog for Eater. However, the extracellular domain of Eater shows resemblance to two scavenger receptors implicated in the removal of apoptotic cells, p120 from flesh fly (Hori, 2000) and CED-1 of C. elegans (Zhou, 2001). In insects, massive clearance of apoptotic cells by macrophages occurs during tissue remodeling in embryogenesis and metamorphosis. In these developmental stages, Eater expression was undetectable at the mRNA level. Consistent with this, transcriptional silencing of eater in S2 cells did not affect the uptake of apoptotic cells in a microscopic assay. Thus, while Eater's expression pattern argues against an important role in the clearance of apoptotic cells, definite assessment of its role in this process will have to await further experimentation (Kocks, 2005).

Drosophila blood cells are generated in two distinct waves of hematopoiesis, embryonic and larval, and both lineages persist throughout larval and adult stages. eater expression was not detected in embryonic and pupal macrophages, while mRNA expression and functional analyses strongly suggest that eater is expressed in larval and adult macrophages. These results indicate that eater expression must be tightly regulated during development. It will be important to confirm this at the protein level and to clarify the lineage relationships between the macrophage subsets that express eater (Kocks, 2005).

No eater expression was detected in lamellocytes and crystal cells, two terminally differentiated, highly specialized immune-defense cell types. Consistent with its lack of expression in lamellocytes, eater is not required for the encapsulation response to parasitic wasp infection. Nor was eater required for humoral immunity in the fly, in agreement with the lack of eater expression in the liver equivalent of the fly (fat body). However, macrophages from eater null animals showed strongly impaired phagocytosis of bacteria, ex vivo and in vivo. These findings point to a specific role for Eater in the control of bacterial infection by phagocytosis (Kocks, 2005).

The role of phagocytosis in Drosophila immunity is less well characterized than the role of the humoral antimicrobial peptide response. This can in part be ascribed to the lack of suitable experimental models, both in terms of Drosophila mutants with impaired phagocyte function and in terms of appropriate infection models. eater null flies were immunocompromised in a gastrointestinal infection model. This phenotype is likely due to decreased phagocytosis of the gastrointestinal pathogen S. marcescens after its escape from the gut lumen and invasion of the body cavity. Thus, eater null flies are a promising tool to address the role of phagocytosis in Drosophila immunity, especially its relative contributions to the NF-kappaB-like pathway-mediated effector mechanisms in this and other infection models. For example, no protective role for eater was seen after septic injury with E. coli or after infection with the fungal pathogen Beauveriana bassiana, consistent with the finding that eater is not required for IMD or Toll pathway signaling (Kocks, 2005).

However, the results indicate that Drosophila phagocytes are able to control the number of pathogenic bacteria that gain access to the hemolymph after crossing the gut epithelium. This is of great interest, as it points to intracellular killing mechanisms in Drosophila phagocytes which are devoid of neutrophil-like granules. It appears that some basic mechanisms of phagocytosis are conserved throughout evolution. The characterization of Eater as a potential microbial phagocytic receptor adds further credence to this idea. It is expected that dissecting the mechanisms by which Eater mediates phagocytosis will be of general interest and might help define the critical steps that are evolutionarily conserved in microbial phagocytosis (Kocks, 2005).

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Ju, J. S., et al. (2006). A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide. J. Immunol. 177: 1838-1845. Medline abstract: 16849495

Kocks, C., et al. (2005). Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123: 335-346. Medline abstract: 16239149

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Vilmos, P., et al. (2004). A rapid rosetting method for separation of hemocyte sub-populations of Drosophila melanogaster. Dev. Comp. Immunol. 28: 555-563. Medline abstract: 15177109

Zettervall, C.-J., et al. (2004). A directed screen for genes involved in Drosophila blood cell activation. Proc. Natl. Acad. Sci. 101: 14192-14197. Medline abstract: 15381778

Zhou, Z., Hartwieg, E. and Horvitz, H. R. (2001). CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell 104: 43-56. Medline abstract: 11163239

date revised: 2 February 2008

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