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Identification of host factors required for cytosol entry by Shigella flexneri: Results of a genome-wide CRISPR/Cas9 screen https://doi.org/10.17863/CAM.102175 O'Donovan, Conor Randow, Felix 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. University of Cambridge 2022-05-09 Thesis Doctoral Doctor of Philosophy (PhD) eng Medical Research Council https://www.repository.cam.ac.uk/handle/1810/358656 https://www.repository.cam.ac.uk/bitstreams/f28be421-0335-4248-af1e-d501159f2fb6/download 52e921da615f1195ce4e2e6c8db220c5 https://www.repository.cam.ac.uk/bitstreams/75ce9913-3af2-4381-9df3-089ff7696e40/download c7a6f910386d9aa951a6d16c63aa838b https://www.repository.cam.ac.uk/bitstreams/b50c99cc-e832-4229-853e-7418e7f06d02/download 16f9c1d2763c46a0c6fc6b50b6a78a1c https://www.repository.cam.ac.uk/bitstreams/49983a8f-0d3c-4282-8924-ca6b71fd1d62/download 26958b4a0106bf9d2737a1b9bb62793b https://www.repository.cam.ac.uk/bitstreams/29bc9444-8432-450e-86f9-c124e2667055/download 8502dc45bae6b9768256ffac2e727e41 https://www.repository.cam.ac.uk/bitstreams/d60082a1-5e34-46db-a750-7c1851ba9d48/download c6e90cd2d8e45af7d7c94f29a0720438 https://www.repository.cam.ac.uk/bitstreams/83a769e0-d821-4bf9-9d95-6f99e9c1c591/download 7516b646c1cd2345f4ca0897eeb17724 https://www.repository.cam.ac.uk/bitstreams/4ad262bf-55d6-43ee-a8cb-afdba3d6165f/download 03be701ef97cf33d1ea22c16dc119c68 https://www.repository.cam.ac.uk/bitstreams/d18a4518-d441-40fb-b0d8-edb9e249c5d3/download 93267e1d783469d27e048405989ecf9f https://www.repository.cam.ac.uk/bitstreams/c275bb49-7520-4c62-8bda-10be627bcc0a/download 2cfb16e35aa7c074a2bf98f08deb9e92 https://www.repository.cam.ac.uk/bitstreams/14ddfe05-5a54-4511-a037-3b9f20c897c6/download e202e82e87ac3cea26a1c803dbd87828 https://www.repository.cam.ac.uk/bitstreams/c1a1b698-40d0-4a60-af1b-345ef2d7f1f5/download c9466f462bd2256fd9d8c461b761288c https://www.repository.cam.ac.uk/bitstreams/4fdc1f08-d9aa-4e30-b719-9c288d1bcc56/download c8e94f9554c72e5ea96bfaa1a7427328 https://www.repository.cam.ac.uk/bitstreams/0dcf0e8f-b2d3-4557-bd4b-ab0994ff3e04/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ bacteria crispr genetic screen immunity pathogen shigella