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

Project description

Finding the factors that control Shigella entrapment in septin cages

The bacterial pathogen Shigella causes bacillary dysentery (shigellosis) and also serves as an important laboratory model organism used to study the host response to infection. Septins are GTP-binding proteins found in all eukaryotic cells except plants. Recent human studies have found that septins build cages around pathogenic bacteria, immobilising and preventing them from invading other cells. The EU-funded CAGECONTROL project aims to discover the factors underlying the entrapment of Shigella in septin cages. Researchers will identify and study new host and bacterial factors behind the formation of septin cages in HeLa cells. The project's results might translate to healthcare applications, inspiring new treatments against shigellosis.

Objective

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.

Coordinator

LONDON SCHOOL OF HYGIENE AND TROPICAL MEDICINE ROYAL CHARTER
Net EU contribution
€ 224 933,76
Address
KEPPEL STREET
WC1E 7HT London
United Kingdom

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Region
London Inner London — West Camden and City of London
Activity type
Higher or Secondary Education Establishments
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Total cost
€ 224 933,76