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Eater: A Big Bite into Phagocytosis

2005; Cell Press; Volume: 123; Issue: 2 Linguagem: Inglês

10.1016/j.cell.2005.10.005

1097-4172

Deniz Ertürk-Hasdemir, Neal Silverman,

Mosquito-borne diseases and control

The phagocytosis of invading microorganisms by specialized blood cells is a crucial element of innate immunity in both mammals and insects. In this issue of Cell, Kocks et al., 2005Kocks C. Cho J.H. Nehme N. Ulvila J. Pearson A.M. Meister M. Strom C. Conto S.L. Hetru C. Stuart L.M. et al.Cell. 2005; 123 (this issue): 335-346Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar demonstrate that Eater, a scavenger receptor, plays an important role in the recognition and phagocytosis of bacteria in the fruit fly Drosophila. The phagocytosis of invading microorganisms by specialized blood cells is a crucial element of innate immunity in both mammals and insects. In this issue of Cell, Kocks et al., 2005Kocks C. Cho J.H. Nehme N. Ulvila J. Pearson A.M. Meister M. Strom C. Conto S.L. Hetru C. Stuart L.M. et al.Cell. 2005; 123 (this issue): 335-346Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar demonstrate that Eater, a scavenger receptor, plays an important role in the recognition and phagocytosis of bacteria in the fruit fly Drosophila. Phagocytosis is an evolutionarily conserved process that is essential for a variety of biological events including the elimination of microorganisms, activation of innate and adaptive immune responses, removal of apoptotic cells, and tissue remodeling during development. Receptor-mediated recognition of infectious microbes is a key early step in phagocytosis. In mammals, phagocytic cells, such as macrophages, express a large number of surface receptors that recognize and engulf microbes. Among these, the Fc receptors, the complement receptors, and the integrins bind to particles opsonized by IgG, complement, and fibronectin/vitronectin, respectively. Scavenger receptors are also important phagocytic receptors that bind to diverse ligands such as acetylated low-density lipoprotein, polyribonucleotides, lipopolysaccharide, as well as whole bacteria (Stuart and Ezekowitz, 2005Stuart L.M. Ezekowitz R.A. Immunity. 2005; 22: 539-550Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). All of these receptors initiate various intracellular signaling pathways to direct the cytoplasmic remodeling required for internalization, phagosome maturation, and the destruction of microbes. In this issue of Cell, Kocks et al., 2005Kocks C. Cho J.H. Nehme N. Ulvila J. Pearson A.M. Meister M. Strom C. Conto S.L. Hetru C. Stuart L.M. et al.Cell. 2005; 123 (this issue): 335-346Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar identify Eater, a scavenger receptor in the fruit fly Drosophila that mediates the phagocytosis of bacterial pathogens. Drosophila has three types of blood cells (hemocytes): crystal cells (which contain enzymes that trigger the phenoloxidase cascade following parasitization or wounding), lamellocytes (which encapsulate large pathogens such as eggs from parasitoid wasps), and plasmatocytes (which are highly phagocytic and comprise the majority of the blood cell population). Plasmatocytes are similar to mammalian macrophages (Meister, 2004Meister M. Curr. Opin. Immunol. 2004; 16: 10-15Crossref PubMed Scopus (124) Google Scholar). Previous studies in insects have identified a number of conserved proteins, including pathogen receptors, complement-like factors, and cytoskeletal proteins that are involved in phagocytosis (Moita et al., 2005Moita L.F. Wang-Sattler R. Michel K. Zimmermann T. Blandin S. Levashina E.A. Kafatos F.C. Immunity. 2005; 23: 65-73Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Pearson et al., 2003Pearson A.M. Baksa K. Ramet M. Protas M. McKee M. Brown D. Ezekowitz R.A. Microbes Infect. 2003; 5: 815-824Crossref PubMed Scopus (102) Google Scholar, Rämet et al., 2001Rämet M. Pearson A. Manfruelli P. Li X. Koziel H. Gobel V. Chung E. Krieger M. Ezekowitz R.A. Immunity. 2001; 15: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). However, the molecular mechanisms of phagocytosis in Drosophila are less well characterized than the antimicrobial peptide response, exemplified by the Toll and the immune deficiency (IMD) signaling pathways, and only a few genes have been implicated in phagocytosis. Using microarray analysis, Kocks and colleagues identified 46 genes that show decreased expression in a Drosophila cell line, called Schneider 2 (S2), following RNAi targeting of Serpent, a transcription factor that is required for phagocytosis and hemocyte differentiation (Rämet et al., 2002Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (564) Google Scholar). Kocks and colleagues targeted each of these genes by RNAi in S2 cells in order to determine their individual roles in phagocytosis. This analysis revealed one gene, which the authors call eater, that has a clear role in the phagocytosis of bacteria, either Escherichia coli or Staphylococcus aureus. RNAi of the eater gene decreased phagocytic activity by 70% relative to the control cells. Consistent with a role in phagocytosis, in Drosophila larva, eater mRNA is predominantly expressed in the plasmatocytes and the lymph glands but not in crystal cells, lamellocytes, or the fatbody, which are cells and organs not involved in phagocytosis. The characterization of mutant flies demonstrates that Eater plays a critical role in phagocytosis in vivo. Phagocytosis of several bacterial species, including S. marcescens and S. aureus, is reduced by up to ∼80% in plasmatocytes from fly larvae that lack Eater. Yet, these same plasmatocytes were unaffected in other hemocyte activities including endocytosis and the phagocytosis of India ink. Similarly, phagocytosis of bacteria was strongly reduced in adult eater mutant flies, and the expression of wild-type Eater in hemocytes partially rescued this phenotype. Moreover, eater mutant flies were hypersusceptible to natural oral infection with S. marcescens. A similar effect is observed in flies in which phagocytosis has been inhibited by other means. eater is predicted to encode a cell-surface receptor with 32 typical EGF-like repeats, which is similar to a scavenger receptor from the flesh fly that has been implicated in tissue remodeling. Interestingly, the characterization of Eater is the first indication that EGF-like repeats are involved in microbial recognition. In vitro, the N-terminal 199 amino acids of Eater, including the N-terminal domain and two of the EGF-like repeats, bind to Gram-negative and Gram-positive bacteria, as well as to yeast. Thus, like other scavenger receptors, Eater appears to recognize a broad range of microbial pathogens. Also typical of scavenger receptors, the binding of Eater to bacteria could be blocked by addition of an excess of acetylated low-density lipoprotein, a scavenger ligand. The loss of Eater has a more dramatic effect on phagocytosis than has been previously observed with mutants in other receptors implicated in phagocytosis. In an earlier study, Rämet et al., 2001Rämet M. Pearson A. Manfruelli P. Li X. Koziel H. Gobel V. Chung E. Krieger M. Ezekowitz R.A. Immunity. 2001; 15: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar identified the scavenger receptor dSR-CI as a pattern-recognition receptor that binds to bacteria but not to yeast. This receptor is responsible for a minor portion (20%–30%) of the total binding of bacteria by S2 cells and is required for optimal phagocytosis. These findings suggested that additional receptors may be involved in phagocytosis. Also, using an RNAi-based screen in S2 cells, Rämet et al., 2002Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (564) Google Scholar reported 34 gene products that are implicated in the phagocytosis of bacteria. Among these genes is the peptidoglycan recognition protein LC, which is involved in the engulfment of Gram-negative, but not Gram-positive, bacteria. Moita et al., 2005Moita L.F. Wang-Sattler R. Michel K. Zimmermann T. Blandin S. Levashina E.A. Kafatos F.C. Immunity. 2005; 23: 65-73Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar reported a similar result for peptidoglycan recognition protein LC (PGRP-LC) in the mosquito Anopheles gambiae. Furthermore, Watson et al., 2005Watson F.L. Puttmann-Holgado R. Thomas F. Lamar D.L. Hughes M. Kondo M. Rebel V.I. Schmucker D. Science. 2005; 309: 1874-1878Crossref PubMed Scopus (511) Google Scholar demonstrated that the immunoglobulin superfamily receptor Dscam (Down syndrome cell adhesion molecule) binds to bacteria and is required for efficient phagocytosis by larval plasmatocytes. Decreased Dscam expression results in ∼30% reduced phagocytosis. Alternative splicing of Dscam potentially produces ∼38,000 isoforms (∼18,000 in fat body cells and hemocytes) with distinct extracellular domains, only some of which bind to E. coli. Therefore, Dscam alone could provide a broad diversity of pathogen recognition in Drosophila, as a cell-surface receptor and/or a secreted, circulating opsonin. Although Eater clearly plays an important role in the phagocytosis of pathogens in Drosophila, many questions still remain unanswered. How do different pattern-recognition receptors physically and functionally interact with each other to effectively control phagocytosis? Simultaneous silencing of the genes dSR-CI, PGRP-LC, and eater decreases phagocytosis of bacteria by S2 cells to a greater extent than silencing of eater alone. This suggests that multiple receptors cooperate in the recognition and engulfment of microbes. These results need to be further tested using biochemical methods and genetic approaches in order to determine whether these multiple receptors are simply redundant or whether they play distinct roles in phagocytosis. For example, phagocytosis involves multiple steps, such as opsonization, binding of microbes, phagosome maturation, phagosome-lysosome fusion, and microbial killing. The question is do these different receptors, each implicated in phagocytosis, control different steps in the process or do they have overlapping and partially redundant functions in microbial recognition? Is the binding of Eater to microorganisms in vivo direct or are there other receptors and co-receptors that cooperate to provide specificity in recognition? Also, Moita et al., 2005Moita L.F. Wang-Sattler R. Michel K. Zimmermann T. Blandin S. Levashina E.A. Kafatos F.C. Immunity. 2005; 23: 65-73Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar have shown that three thio-ester containing complement-related proteins from mosquito—TEP1, TEP3, and TEP4—are required for efficient phagocytosis of bacteria. Similar to mammalian complement factors, TEP1 in A. gambiae opsonizes bacteria via a thio-ester bond (Levashina et al., 2001Levashina E.A. Moita L.F. Blandin S. Vriend G. Lagueux M. Kafatos F.C. Cell. 2001; 104: 709-718Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The Drosophila genome also encodes six TEP proteins that have recently been linked to phagocytosis, with certain TEPs being required for the phagocytosis of distinct classes of microbes (Stroschein-Stevenson, Foley, O’Farrell, and Johnson, personal communication). It will be interesting to learn how opsonins, such as the TEPs and secreted Dscam isoforms, interact with the various cell-surface receptors involved in phagocytosis in insects. Another question is how these cell-surface receptors regulate the intracellular pathways that direct cytoskeletal remodeling and membrane trafficking, which are required for internalization and digestion of microbes? And finally, is phagocytosis coupled to the antimicrobial immune response? Although PGRP-LC activates the IMD pathway (Choe et al., 2002Choe K.M. Werner T. Stoven S. Hultmark D. Anderson K.V. Science. 2002; 296: 359-362Crossref PubMed Scopus (452) Google Scholar, Gottar et al., 2002Gottar M. Gobert V. Michel T. Belvin M. Duyk G. Hoffmann J.A. Ferrandon D. Royet J. Nature. 2002; 416: 640-644Crossref PubMed Scopus (491) Google Scholar) in addition to its minor role in phagocytosis (Rämet et al., 2002Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (564) Google Scholar), eater mutant flies respond normally to stimulation of the IMD and Toll signaling pathways (Kocks et al., 2005Kocks C. Cho J.H. Nehme N. Ulvila J. Pearson A.M. Meister M. Strom C. Conto S.L. Hetru C. Stuart L.M. et al.Cell. 2005; 123 (this issue): 335-346Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). This suggests that Eater is not involved in antimicrobial peptide signaling pathways in insects. However, it is not clear whether other factors may coordinately regulate antimicrobial peptide gene expression and phagocytosis. Given the powerful genetic and molecular tools that can be used in Drosophila, it is likely that more players in phagocytosis will be discovered and that many of these questions will be answered in the near future.