Abstract
The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages1,2, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rizki, T. M. & Rizki, R. M. Properties of the larval hemocytes of Drosophila melanogaster. Experimentia 36, 1223–1226 (1980)
Franc, N. C., Heitzler, P., Ezekowitz, R. A. & White, K. Requirement for Croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284, 1991–1994 (1999)
Aderem, A. & Underhill, D. M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17, 593–623 (1999)
Garin, J. et al. The phagosome proteome: insight into phagosome functions. J. Cell. Biol. 152, 165–180 (2001)
Rämet, M. et al. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15, 1027–1038 (2001)
Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000)
Pearson, A., Lux, A. & Krieger, M. Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster. Proc. Natl Acad. Sci. USA 92, 4056–4060 (1995)
Bjerknes, R. Flow cytometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans. J. Immunol. Methods 72, 229–241 (1984)
Kirchhausen, T. Three ways to make a vesicle. Nature Rev. Mol. Cell. Biol. 1, 187–198 (2000)
Hackam, D. J. et al. Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis. J. Biol. Chem. 276, 18200–18208 (2001)
Perry, D. G., Daugherty, G. L. & Martin, W. J. II Clathrin-coated pit-associated proteins are required for alveolar macrophage phagocytosis. J. Immunol. 162, 380–386 (1999)
Lebestky, T., Chang, T., Hartenstein, V. & Banerjee, U. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science 288, 146–149 (2000)
Rehorn, K. P., Thelen, H., Michelson, A. M. & Reuter, R. A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development 122, 4023–4031 (1996)
Oike, Y. et al. Mice homozygous for a truncated form of CREB-binding protein exhibit defects in hematopoiesis and vasculo-angiogenesis. Blood 93, 2771–2779 (1999)
Fehon, R. G., Dawson, I. A. & Artavanis-Tsakonas, S. A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development 120, 545–557 (1994)
Lee, S., Harris, K. L., Whitington, P. M. & Kolodziej, P. A short stop is allelic to kakapo, and encodes rod-like cytoskeletal-associated proteins required for axon extension. J. Neurosci. 20, 1096–1108 (2000)
Madden, K., Crowner, D. & Giniger, E. LOLA has the properties of a master regulator of axon-target interaction for SNb motor axons of Drosophila. Dev. Biol. 213, 301–313 (1999)
Juang, J. L. & Hoffmann, F. M. Drosophila Abelson interacting protein (dAbi) is a positive regulator of Abelson tyrosine kinase activity. Oncogene 18, 5138–5147 (1999)
Lanzetti, L. et al. The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408, 374–377 (2000)
Werner, T. et al. A family of Peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Proc. Natl Acad. Sci. USA 97, 13772–13777 (2000)
Kang, D., Liu, G., Lundstrom, A., Gelius, E. & Steiner, H. A Peptidoglycan recognition protein in innate immunity conserved from insects to humans. Proc. Natl Acad. Sci. USA 95, 10078–10082 (1998)
Ochiai, M. & Ashida, M. A pattern recognition protein for peptidoglycan. Cloning the cDNA and the gene of the silkworm, Bombyx mori. J. Biol. Chem. 274, 11854–11858 (1999)
Liu, C., Gelius, E., Liu, G., Steiner, H. & Dziarski, R. Mammalian Peptidoglycan recognition protein binds peptidoglycan with high affinity, is expressed in neutrophils, and inhibits bacterial growth. J. Biol. Chem. 275, 24490–24499 (2000)
Liu, C., Xu, Z., Gupta, D. & Dziarski, R. Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules. J. Biol. Chem. 276, 34686–34694 (2001)
Michel, T., Reichhart, J. M., Hoffmann, J. A. & Royet, J. Drosophila Toll is activated by Gram-positive bacteria through a circulating Peptidoglycan recognition protein. Nature 414, 756–759 (2001)
Tauszig, S., Jouanguy, E., Hoffmann, J. A. & Imler, J. L. Toll-related receptors and the control of antimicrobial peptide expression in Drosophila. Proc. Natl Acad. Sci. USA 97, 10520–10525 (2000)
Imler, J. L., Tauszig, S., Jouanguy, E., Forestier, C. & Hoffmann, J. A. LPS-induced immune response in Drosophila. J. Endotoxin Res. 6, 459–462 (2000)
Lukacsovich, T. et al. Dual-tagging gene trap of novel genes in Drosophila melanogaster. Genetics 157, 727–742 (2001)
Imler, J. L. & Hoffmann, J. A. Signaling mechanisms in the antibacterial host defense of Drosophila. Curr. Opin. Microbiol. 3, 16–22 (2000)
Leulier, F., Rodriguez, A., Khush, R. S., Abrams, J. M. & Lemaitre, B. The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep. 1, 353–358 (2000)
Manfruelli, P., Reichhart, J. M., Steward, R., Hoffmann, J. A. & Lemaitre, B. A mosaic analysis in Drosophila fat body cells of the control of antimicrobial peptide genes by the Rel proteins Dorsal and DIF. EMBO J. 18, 3380–3391 (1999)
Acknowledgements
M.R. devised and carried out the RNAi screen and performed microarray and luciferase analysis. P.M. did the in vivo fly work under supervision of B.M.-P. A.P. did some of the targeted RNAi treatments and was intellectually involved throughout the project. M.R. and R.A.B.E. co-wrote the paper. We thank members of Hoffmann and Ezekowitz laboratories for discussions. We also thank M. Sackal for help with northern blots, J. Couget and M. Tahiliani for help with Affymetrix microrrays. M. Krieger provided the S2 cell cDNA library. This work was supported by grants from the National Institutes of Health (R.A.B.E.), the Foundation for Pediatric Research (M.R.), the Finnish Medical Foundation (M.R.), Maud Kuistila Foundation (M.R.), l'A.R.C. (P.M.) and American Cancer Society (A.P.).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare that they have no competing financial interests
Supplementary information
Rights and permissions
About this article
Cite this article
Rämet, M., Manfruelli, P., Pearson, A. et al. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416, 644–648 (2002). https://doi.org/10.1038/nature735
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature735
This article is cited by
-
Drosophila caspases as guardians of host-microbe interactions
Cell Death & Differentiation (2023)
-
Localized efficacy of environmental RNAi in Tetranychus urticae
Scientific Reports (2022)
-
Characterization of PGRP-LB and immune deficiency in the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae)
Applied Entomology and Zoology (2022)
-
Sensing microbial infections in the Drosophila melanogaster genetic model organism
Immunogenetics (2022)
-
A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells
Scientific Reports (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.