Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium’s adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled S. Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.
We are very grateful to Leigh Knodler for her generous gift of P22 phages from a S. Typhimurium glmS::Cm::mCherry strain. We thank the Microscopy and Histology Core Facility, the Centre for Genome-Enabled Biology and Medicine (CGEBM), the Iain Fraser Cytometry Centre and the qPCR Facility (University of Aberdeen) for their support and assistance in this work. We thank members of the Spanò/Baldassarre laboratory for their feedback throughout this project. The content of this manuscript has been posted as a preprint on bioRxiv (Solano-Collado et al., 2020).
This work was supported by the European Union’s Horizon 2020 research and innovation program Marie Skłodowska-Curie Fellowship (706040_KILLINGTYPHI) to VS-C, the Wellcome Trust (Seed Award 109680/Z/15/Z), the European Union’s Horizon 2020 ERC consolidator award (2016-726152-TYPHI), the BBSRC (BB/N017854/1) and Tenovus Scotland (G14/19) to SS.
- Salmonella Typhi
- Rab GTPases
- shRNA screen