{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/358656"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/358656","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Identification of host factors required for cytosol entry by Shigella flexneri: Results of a genome-wide CRISPR/Cas9 screen","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["O'Donovan, Conor"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Randow, Felix"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-05-09","date_published":"2022-05-09","updated_at":"2026-07-22T22:23:56Z","subjects":["bacteria","crispr","genetic screen","immunity","pathogen","shigella"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/0dcf0e8f-b2d3-4557-bd4b-ab0994ff3e04/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.102175","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Randow, Felix"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Medical Research Council"]},{"key":"dc:creator","label":"Author","values":["O'Donovan, Conor"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-05-09"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/358656"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["bacteria","crispr","genetic screen","immunity","pathogen","shigella"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/0dcf0e8f-b2d3-4557-bd4b-ab0994ff3e04/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.102175"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/f28be421-0335-4248-af1e-d501159f2fb6/download","https://www.repository.cam.ac.uk/bitstreams/75ce9913-3af2-4381-9df3-089ff7696e40/download","https://www.repository.cam.ac.uk/bitstreams/b50c99cc-e832-4229-853e-7418e7f06d02/download","https://www.repository.cam.ac.uk/bitstreams/49983a8f-0d3c-4282-8924-ca6b71fd1d62/download","https://www.repository.cam.ac.uk/bitstreams/29bc9444-8432-450e-86f9-c124e2667055/download","https://www.repository.cam.ac.uk/bitstreams/d60082a1-5e34-46db-a750-7c1851ba9d48/download","https://www.repository.cam.ac.uk/bitstreams/83a769e0-d821-4bf9-9d95-6f99e9c1c591/download","https://www.repository.cam.ac.uk/bitstreams/4ad262bf-55d6-43ee-a8cb-afdba3d6165f/download","https://www.repository.cam.ac.uk/bitstreams/d18a4518-d441-40fb-b0d8-edb9e249c5d3/download","https://www.repository.cam.ac.uk/bitstreams/c275bb49-7520-4c62-8bda-10be627bcc0a/download","https://www.repository.cam.ac.uk/bitstreams/14ddfe05-5a54-4511-a037-3b9f20c897c6/download","https://www.repository.cam.ac.uk/bitstreams/c1a1b698-40d0-4a60-af1b-345ef2d7f1f5/download","https://www.repository.cam.ac.uk/bitstreams/4fdc1f08-d9aa-4e30-b719-9c288d1bcc56/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. 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