The Gram-negative bacterial pathogen Shigella flexneri remains a globally significant cause of morbidity and mortality. Shigella induces its uptake into colonic epithelial cells via a short-lived vacuole, and lyses the vacuole membrane to gain entry to the host cytosol. As a professional cytosol-dwelling pathogen, Shigella then overcomes cell-autonomous host defences to establish its replicative niche, before spreading into adjacent enterocytes. Although cytosol entry is a key step in its cellular pathogenesis, the mechanisms by which Shigella achieves this are not fully understood.

To identify essential host factors and key pathways required by Shigella in the early stages of its pathogenesis, I developed and carried out a genome-wide CRISPR/Cas9 knockout screen using a tissue culture model of epithelial cell infection. I used a hierarchical validation method which combined arrayed high-content microscopy and semi-automated image analysis to confirm a novel set of host factors which facilitate Shigella infection. I then established in greater detail the roles of three of the major identified hits, using a further panel of microscopy, flow cytometry and intracellular replication assays. I showed that the multifunctional protein KHDRBS1, known to act as a signalling adaptor and regulator of inflammatory responses, facilitates cytosol entry and replication by Shigella. I showed that the transcriptional regulator YY1 is also required for cytosol entry and replication. Both KHDRBS1 and YY1 are likely to act indirectly in Shigella infection by their impact on host processes. The adaptor protein (AP)-3 subunit AP3M2 is involved in protein sorting in the post-Golgi endosomal network. I showed that AP3M2 is required for Shigella invasion and cytosol entry, and that it enriches transiently in the vicinity of the bacterial entry focus. Lastly, I found that AP3M2 is also targeted by Shigella for degradation in a host proteasome- dependent manner, along with additional subunits of the AP-2 and AP-3 complexes.

Overall, this work identifies and characterises three novel host factors required for the cellular pathogenesis of Shigella infection, and provides a dataset that better informs our understanding of the host-Shigella interface. These findings complement current models of invasion and vacuole rupture by Shigella, and will help to direct the rational development of improved vaccination and therapeutic strategies.