Source: http://www.asmscience.org/content/book/10.1128/9781555816650.ch35
Timestamp: 2019-04-25 06:19:21+00:00

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The development of whole genome RNA interference (RNAi) by using double-stranded RNA (dsRNA) in Drosophila 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 Drosophila macrophages. Coupled with the fact that Drosophila 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 Drosophila 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.
Schematic outline of different phenotypes in fluorescent-based macrophage-pathogen screens. (A) L. monocytogenes ( Agaisse et al., 2005 ): (i) normal phagocytosis by S2 cells; (ii) decreased phagocytosis after RNAi; (iii) “spot” phenotype (see text); (iv) increased bacterial viability within S2 cell. (B) C. albicans ( Stroschein-Stevenson et al., 2006 ). The screen utilized GFP-expressing Candida. Nonphagocytosed cells were distinguished from phagocytosed yeast by secondary staining (thick outline) with an anti-Candida antibody. (C) M. fortuitum ( Philips et al., 2005 ). 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).
Schematic showing pathogen-specific factors in S2 cells identified by RNAi and other screens.
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