Genome-Wide RNAi Screen for Host Factors Required for Intracellular Bacterial Infection

Abstract

Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

Methods to study Drosophila immunity

“…While the patchy efficiency of first generation RNAis relegated in vivo screens to fishing expeditions, improved second-generation RNAi libraries promise optimized throughput in RNAi-based screens. S2-based RNAi screens have successfully identified novel Toll, Imd and JNK pathway components [56][57][58][59], determinants of intracellular pathogen resistance [60] or phagocytic receptors on hemocytes [61,62], and in vivo RNAi screens have contributed to our understanding of Drosophila hemocyte development or response to intestinal infection [63,64].…”

Genome-wide RNAi screen of Ca 2+ influx identifies genes that regulate Ca 2+ release-activated Ca 2+ channel activity

“…It was neither a hit in a previous screen of cell survival (9) nor in any other published Drosophila whole-genome RNAi screen (10)(11)(12)(13)(14). The olf186-F gene is a member of a highly conserved gene family that contains three homologs in mammals, two in chicken, three in zebrafish, and one member only in fly and worm (see Fig.…”

“…To search systematically for additional components of the CRAC channel, and to analyze the signaling network and other required factors that lead to SOC channel activity, we devised and performed a genome-wide screen on S2 cells based on a fluorescence assay of Ca 2ϩ influx. The library at Harvard's Drosophila RNAi Screening Center (DRSC) of 23,845 dsRNA amplicons has been used in several functional screens (9)(10)(11)(12)(13)(14).A very recent report identified a genetic defect in patients with severe combined immune deficiency (SCID) (15). The screen in this study made use of the ability of thapsigargin (TG) to send GFP-tagged nuclear factor of activated T cells (NFAT) to the nucleus in S2 cells, providing an assay for disruption of signaling anywhere in the cascade from elevated [Ca 2ϩ ] i to calcineurin activation and nuclear relocalization of NFAT.…”

“…To search systematically for additional components of the CRAC channel, and to analyze the signaling network and other required factors that lead to SOC channel activity, we devised and performed a genome-wide screen on S2 cells based on a fluorescence assay of Ca 2ϩ influx. The library at Harvard's Drosophila RNAi Screening Center (DRSC) of 23,845 dsRNA amplicons has been used in several functional screens (9)(10)(11)(12)(13)(14).…”

Functional high-throughput screening identifies the miR-15 microRNA family as cellular restriction factors for Salmonella infection

“…The use of RNA interference screenings has improved our knowledge of the interplay between bacteria and host cells, identifying novel cellular factors critical for infection [17][18][19] . Only recently this approach has been extended to miRNAs 20,21 , and is yet to be applied for the identification of miRNAs critical for infection by bacterial pathogens.…”