Chapter 35 : Whole Genome Screens in Macrophages

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The development of whole genome RNA interference (RNAi) by using double-stranded RNA (dsRNA) in cells opened up the potential to screen the entire genome of macrophage-like cells for host proteins that modulate bacterial infection. This chapter focuses on how these screens have been used in in vitro studies with several different pathogens, both intra- and extracellular. The S2 cell line is thought to be derived from embryonic plasmatocytes and behaves similarly to primary macrophages. Coupled with the fact that lack the interferon response, allowing use of (relatively) inexpensive dsRNAs (unlike mammalian cells), and that S2 cells take up dsRNAs passively through the receptor Eater, it is reasonable that whole genome screens for macrophage function using RNAi first became viable in cell lines. A remarkably small group of genes appear to be required for phagocytosis of all the pathogens studied so far, and most of these are concerned with actin remodeling and vesicle trafficking. The power and usefulness of genome-wide screens utilizing RNAi technology depends on the critical assumption that gene silencing is a very specific process.

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
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Image of FIGURE 1

Schematic outline of different phenotypes in fluorescent-based macrophage-pathogen screens. (A) ( ): (i) normal phagocytosis by S2 cells; (ii) decreased phagocytosis after RNAi; (iii) “spot” phenotype (see text); (iv) increased bacterial viability within S2 cell. (B) ( ). The screen utilized GFP-expressing . Nonphagocytosed cells were distinguished from phagocytosed yeast by secondary staining (thick outline) with an anti- antibody. (C) ( ). This screen utilized GFP under a macrophage-activated promoter in the bacteria (i). Non-fluorescence could signify nonphagocytosed cells (ii) or phagosome-vacuolar escape (iii).

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
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Image of FIGURE 2

Schematic showing pathogen-specific factors in S2 cells identified by RNAi and other screens.

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
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1. Adams, M. D.,, S. E. Celniker,, R. A. Holt,, C. A. Evans,, J. D. Gocayne,, P. G. Amanatides,, S. E. Scherer,, P. W. Li,, R. A. Hoskins,, R. F. Galle,, R. A. George,, S. E. Lewis,, S. Richards,, M. Ashburner,, S. N. Henderson,, G. G. Sutton,, J. R. Wortman,, M. D. Yandell,, Q. Zhang,, L. X. Chen,, R. C. Brandon,, Y. H. Rogers,, R. G. Blazej,, M. Champe,, B. D. Pfeiffer,, K. H. Wan,, C. Doyle,, E. G. Baxter,, G. Helt,, C. R. Nelson,, G. L. Gabor,, J. F. Abril,, A. Agbayani,, H. J. An,, C. Andrews-Pfannkoch,, D. Baldwin,, R. M. Ballew,, A. Basu,, J. Baxendale,, L. Bayraktaroglu,, E. M. Beasley,, K. Y. Beeson,, P. V. Benos,, B. P. Berman,, D. Bhandari,, S. Bolshakov,, D. Borkova,, M. R. Botchan,, J. Bouck,, P. Brokstein,, P. Brottier,, K. C. Burtis,, D. A. Busam,, H. Butler,, E. Cadieu,, A. Center,, I. Chandra,, J. M. Cherry,, S. Cawley,, C. Dahlke,, L. B. Davenport,, P. Davies,, B. de Pablos,, A. Delcher,, Z. Deng,, A. D. Mays,, I. Dew,, S. M. Dietz,, K. Dodson,, L. E. Doup,, M. Downes,, S. Dugan-Rocha,, B. C. Dunkov,, P. Dunn,, K. J. Durbin,, C. C. Evangelista,, C. Ferraz,, S. Ferriera,, W. Fleischmann,, C. Fosler,, A. E. Gabrielian,, N. S. Garg,, W. M. Gelbart,, K. Glasser,, A. Glodek,, F. Gong,, J. H. Gorrell,, Z. Gu,, P. Guan,, M. Harris,, N. L. Harris,, D. Harvey,, T. J. Heiman,, J. R. Hernandez,, J. Houck,, D. Hostin,, K. A. Houston,, T. J. Howland,, M. H. Wei,, C. Ibegwam,, M. Jalali,, F. Kalush,, G. H. Karpen,, Z. Ke,, J. A. Kennison,, K. A. Ketchum,, B. E. Kimmel,, C. D. Kodira,, C. Kraft,, S. Kravitz,, D. Kulp,, Z. Lai,, P. Lasko,, Y. Lei,, A. A. Levitsky,, J. Li,, Z. Li,, Y. Liang,, X. Lin,, X. Liu,, B. Mattei,, T. C. McIntosh,, M. P. McLeod,, D. McPherson,, G. Merkulov,, N. V. Milshina,, C. Mobarry,, J. Morris,, A. Moshrefi,, S. M. Mount,, M. Moy,, B. Murphy,, L. Murphy,, D. M. Muzny,, D. L. Nelson,, D. R. Nelson,, K. A. Nelson,, K. Nixon,, D. R. Nusskern,, J. M. Pacleb,, M. Palazzolo,, G. S. Pittman,, S. Pan,, J. Pollard,, V. Puri,, M. G. Reese,, K. Reinert,, K. Remington,, R. D. Saunders,, F. Scheeler,, H. Shen,, B. C. Shue,, I. Siden-Kiamos,, M. Simpson,, M. P. Skupski,, T. Smith,, E. Spier,, A. C. Spradling,, M. Stapleton,, R. Strong,, E. Sun,, R. Svirskas,, C. Tector,, R. Turner,, E. Venter,, A. H. Wang,, X. Wang,, Z. Y. Wang,, D. A. Wassarman,, G. M. Weinstock,, J. Weissenbach,, S. M. Williams,, T. Woodage,, K. C. Worley,, D. Wu,, S. Yang,, Q. A. Yao,, J. Ye,, R. F. Yeh,, J. S. Zaveri,, M. Zhan,, G. Zhang,, Q. Zhao,, L. Zheng,, X. H. Zheng,, F. N. Zhong,, W. Zhong,, X. Zhou,, S. Zhu,, X. Zhu,, H. O. Smith,, R. A. Gibbs,, E. W. Myers,, G. M. Rubin, and, J. C. Venter. 2000. The genome sequence of Drosophila melanogaster. Science 287: 21852195.
2. Agaisse, H.,, L. S. Burrack,, J. A. Philips,, E. J. Rubin,, N. Perrimon, and, D. E. Higgins. 2005. Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science 309: 12481251.
3. Apidianakis, Y.,, M. N. Mindrinos,, W. Xiao,, G. W. Lau,, R. L. Baldini,, R. W. Davis, and, L. G. Rahme. 2005. Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression. Proc. Natl. Acad. Sci. USA 102: 25732578.
4. Bernards, R.,, T. R. Brummelkamp, and, R. L. Beijersbergen. 2006. shRNA libraries and their use in cancer genetics. Nat. Methods 3: 701706.
5. Boutros, M.,, A. A. Kiger,, S. Armknecht,, K. Kerr,, M. Hild,, B. Koch,, S. A. Haas,, R. Paro, and, N. Perrimon. 2004. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303: 832835.
6. Cheng, L. W.,, J. P. Viala,, N. Stuurman,, U. Wiedemann,, R. D. Vale, and, D. A. Portnoy. 2005. Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen. Proc. Natl. Acad. Sci. USA 102: 1364603651.
7. Choe, K. M.,, H. Lee, and, K. V. Anderson. 2005. Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. USA 102: 11221126.
8. Clemens, J. C.,, C. A. Worby,, N. Simonson-Leff,, M. Muda,, T. Maehama,, B. A. Hemmings, and, J. E. Dixon. 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97: 64996503.
9. de Hostos, E. L.,, C. Rehfuess,, B. Bradtke,, D. R. Waddell,, R. Albrecht,, J. Murphy, and, G. Gerisch. 1993. Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility. J. Cell Biol. 120: 163173.
10. Dietrich, W. F. 2001. Using mouse genetics to understand infectious disease pathogenesis. Genome Res. 11: 325331.
11. Dorer, M. S.,, D. Kirton,, J. S. Bader, and, R. R. Isberg. 2006. RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics. PLoS Pathog. 2: e34.
12. Echeverri, C. J.,, P. A. Beachy,, B. Baum,, M. Boutros,, F. Buchholz,, S. K. Chanda,, J. Downward,, J. Ellenberg,, A. G. Fraser,, N. Hacohen,, W. C. Hahn,, A. L. Jackson,, A. Kiger,, P. S. Linsley,, L. Lum,, Y. Ma,, B. Mathey-Prevot,, D. E. Root,, D. M. Sabatini,, J. Taipale,, N. Perrimon, and, R. Bernards. 2006. Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3: 777779.
13. Echeverri, C. J.,, and N. Perrimon. 2006. High-throughput RNAi screening in cultured cells: a user’s guide. Nat. Rev. Genet. 7: 373384.
14. Foley, E.,, and P. H. O’Farrell. 2004. Functional dissection of an innate immune response by a genome-wide RNAi screen. PLoS Biol. 2: E203.
15. Franc, N. C.,, P. Heitzler,, R. A. Ezekowitz, and, K. White. 1999. Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284: 19911994.
16. Garver, L. S.,, J. Wu, and, L. P. Wu. 2006. The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila. Proc. Natl. Acad. Sci. USA 103: 660665.
17. Hammond, S. M.,, E. Bernstein,, D. Beach, and, G. J. Hannon. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293296.
18. Ichikawa, J. K.,, S. B. English,, M. C. Wolfgang,, R. Jackson,, A. J. Butte, and, S. Lory. 2005. Genome-wide analysis of host responses to the Pseudomonas aeruginosa type III secretion system yields synergistic effects. Cell. Microbiol. 7: 16351646.
19. Jackson, A. L.,, S. R. Bartz,, J. Schelter,, S. V. Kobayashi,, J. Burchard,, M. Mao,, B. Li,, G. Cavet, and, P. S. Linsley. 2003. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21: 635637.
20. Kaneko, T.,, T. Yano,, K. Aggarwal,, J. H. Lim,, K. Ueda,, Y. Oshima,, C. Peach,, D. Erturk-Hasdemir,, W. E. Goldman,, B. H. Oh,, S. Kurata, and, N. Silverman. 2006. PGRP-LC and PGRP-LE have essential yet distinct functions in the drosophila immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 7: 715723.
21. Kocks, C.,, J. H. Cho,, N. Nehme,, J. Ulvila,, A. M. Pearson,, M. Meister,, C. Strom,, S. L. Conto,, C. Hetru,, L. M. Stuart,, T. Stehle,, J. A. Hoffmann,, J. M. Reichhart,, D. Ferrandon,, M. Ramet, and, R. A. Ezekowitz. 2005. Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123: 335346.
22. Kramnik, I.,, and V. Boyartchuk. 2002. Immunity to intracellular pathogens as a complex genetic trait. Curr. Opin. Microbiol. 5: 111117.
23. Kulkarni, M. M.,, M. Booker,, S. J. Silver,, A. Friedman,, P. Hong,, N. Perrimon, and, B. Mathey-Prevot. 2006. Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3: 833838.
24. Li, Z.,, J. M. Solomon, and, R. R. Isberg. 2005. Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole. Cell. Microbiol. 7: 431442.
25. Maniak, M.,, R. Rauchenberger,, R. Albrecht,, J. Murphy, and, G. Gerisch. 1995. Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell 83: 915924.
26. Nau, G. J.,, J. F. Richmond,, A. Schlesinger,, E. G. Jennings,, E. S. Lander, and, R. A. Young. 2002. Human macrophage activation programs induced by bacterial pathogens. Proc. Natl. Acad. Sci. USA 99: 15031508.
27. Perrimon, N.,, and B. Mathey-Prevot. 2007. Applications of high-throughput RNA interference screens to problems in cell and developmental biology. Genetics 175: 716.
28. Philips, J. A.,, M. C. Porto,, H. Wang,, E. J. Rubin, and, N. Perrimon. 2008. ESCRT factors restrict mycobacterial growth. Proc. Natl. Acad. Sci. USA 105: 30703075.
29. Philips, J. A.,, E. J. Rubin, and, N. Perrimon. 2005. Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309: 12511253.
30. Ramakrishnan, L.,, N. A. Federspiel, and, S. Falkow. 2000. Granuloma-specific expression of Mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science 288: 14361439.
31. Ramet, M.,, A. Pearson,, P. Manfruelli,, X. Li,, H. Koziel,, V. Gobel,, E. Chung,, M. Krieger, and, R. A. Ezekowitz. 2001. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15: 10271038.
32. Rizki, R. M.,, and T. M. Rizki. 1990. Parasitoid virus-like particles destroy Drosophila cellular immunity. Proc. Natl. Acad. Sci. USA 87: 83888392.
33. Rytkonen, A.,, and D. W. Holden. 2007. Bacterial interference of ubiquitination and deubiquitination. Cell Host Microbe 1: 1322.
34. Stroschein-Stevenson, S. L.,, E. Foley,, P. H. O’Farrell, and, A. D. Johnson. 2006. Identification of Drosophila gene products required for phagocytosis of Candida albicans. PLoS Biol. 4: e4.
35. Ulvila, J.,, M. Parikka,, A. Kleino,, R. Sormunen,, R. A. Ezekowitz,, C. Kocks, and, M. Ramet. 2006. Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J. Biol. Chem. 281: 1437014375.


Generic image for table

Genes implicated in phagocytosis by at least three independent screens

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35
Generic image for table

Summary of genome-wide phagocytosis screens

Citation: Javid B, Rubin E. 2009. Whole Genome Screens in Macrophages, p 537-543. In Russell D, Gordon S (ed), Phagocyte-Pathogen Interactions. ASM Press, Washington, DC. doi: 10.1128/9781555816650.ch35

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