Human Genome-Wide RNAi Screen for Host Factors That Modulate Intracellular Salmonella Growth

Abstract

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.

Human Xenobiotic Nuclear Receptor PXR AugmentsMycobacterium tuberculosisSurvival

“…A number of host targets have been identified that are known to modulate host microbicidal properties (11)(12)(13)(14)(15)(16). Among the host factors, NRs in particular have been shown to modulate host effector functions that are imperative for clearance or survival of intracellular bacteria and parasites, including Listeria monocytogenes, Salmonella typhimurium, Leishmania donovani, and M. tuberculosis (17)(18)(19)(20)(21).…”

Identification of host factors required for Yersinia pestis macrophage intracellular survival and their impact on vacuole maturation, acidification and trafficking.

“…Furthermore, inhibition of Rab1 by either RNAi or expression of dominant negative Rab1 constructs indicate that Rab1 function is required for the survival/growth of L. pneumophila [189], C. burnetii [188], S. enterica Typhimurium [133,192], and Brucella melitensis [138]. Our data demonstrate for the first time that Y. pestis also belongs to this group.…”

“…To date, there have been multiple genome-wide RNAi high-throughput screens to identify novel targets and pathways exploited by various bacterial and viral pathogens (Table 1-1; [130][131][132][133][134][135][136][137][138][139][140][141][142]). …”

“…From here, the user can quantify a range of phenotypes based on the amount of fluorescent reporter being imaged. A basic example of this for RNAi pathogen screens is number of bacteria or bacterial total intensity per cell [133,135,141]. To date, there has not been a multiplexed approach to determine both pathogen survival and relationship to either an overexpressed protein or antibody stained protein.…”

“…RNAi has been used to identify host factors required for intracellular survival of several pathogens [130][131][132][133][135][136][137][138][139][140][141][142]. However, most of these screens have used Drosophila or HeLa cell lines, which may not present the same hurdles to intracellular survival as a pathogen may encounter during infection of macrophages.…”