Identification of host factors required for cytosol entry by Shigella flexneri: Results of a genome-wide CRISPR/Cas9 screen

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• O'Donovan, Conor

Advisor dc:contributor.advisor

• Randow, Felix

• bacteria
• crispr
• genetic screen
• immunity
• pathogen
• shigella

Licence

• https://www.repository.cam.ac.uk/bitstreams/0dcf0e8f-b2d3-4557-bd4b-ab0994ff3e04/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.102175

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/358656