Identification and manipulation of factors that control Shigella flexneri entrapment in septin cages.

Shigella is a Gram-negative bacterial pathogen and causative agent of bacillary dysentery (also called shigellosis). Shigella kills ∼164 thousand people per year and is recognised by the WHO as a priority pathogen due to the emergence of antibiotic resistant strains. In the laboratory, Shigella is an important model organism used to study cellular microbiology and host response to infection. This proposal will innovate approaches to discover factors underlying the entrapment of Shigella in "septin cages", a host defence mechanism discovered by Prof. Mostowy. Septins are highly conserved GTP-binding proteins that polymerize into cage-like structures to entrap actively dividing intracellular Shigella, preventing their division and actin-based motility. Here, I will identify and study new host and bacterial factors underlying the formation of septin cages in HeLa cells. For this, I will use a proximity biotinylation assay (split APEX2) specifically localized at sites of Shigella-septin association, followed by mass spectrometry. I will then investigate the identified candidates by siRNA and CRISPR/Cas9 depletion in the case of the host, and by homologous recombination in the case of the bacteria, to monitor the number, morphology and dynamics of septin cages using high-content microscopy. In addition, I will do GFP-fusions to analyse the subcellular localisation of the candidates by high-resolution microscopy. Finally, to control the fate of intracellular Shigella, I will manipulate septin cage formation using nanobodies to artificially deliver septins (and other septin caging factors identified by mass spectrometry) to the surface of Shigella during infection. It is expected that my research will discover fundamental mechanisms underlying the entrapment of Shigella in septin cages, and illuminate issues central to both cell and infection biology. Moreover, my project may translate to human health impact, inspiring new treatments against shigellosis.