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Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli

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.

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Figure 1: Quantification of phagocytosis of E. coli (a, c, e) and S. aureus (b, d, f) by FACS. S2 cells were incubated with FITC-labelled bacteria for 30 min at 4 °C followed by 15 (E. coli) or 20 (S. aureus) min incubation at 26 °C to allow internalization.
Figure 2: PGRP-LC RNAi decreases phagocytosis of E. coli but does not affect phagocytoisis of S. aureus.
Figure 3: PGRP-LC controls Imd pathway-mediated response to Gram-negative bacteria both in vitro and in vivo.

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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.).

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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

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