CAGECONTROL · Identification and manipulation of factors that control Shigella flexneri entrapment in septin cages.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2021-02-01 → 2023-01-31
- EU contribution
- €224,934
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Identification and manipulation of factors that control Shigella flexneri entrapment in septin cages.
Shigella flexneri is an important human pathogen responsible for more than 150 thousand deaths a year in low and middle-income countries and is highlighted as an urgent health threat by the WHO due to the lack of an effective vaccine and the emergence of antibiotic resistant strains. In addition to its clinical importance, the study of S. flexneri benefits from a great experimental toolbox due to its genetic proximity to Escherichia coli. For this reason and after more than 30 years of fundamental research, S. flexneri has become a paradigm to study intracellular infection. This means that many of the fundamental mechanisms of bacterial pathogenicity and host response discovered with S. flexneri can also be applied to other important human pathogens. In particular, research on S. flexneri has contributed to the discovery of multiple cell autonomous immune mechanisms that hold great promise for human health impact. One of these cell autonomous immune responses is controlled by septins, a poorly studied component of the host cytoskeleton. In 2010, septins were shown to entrap intracellular S. flexneri in cage-like structures, which prevent their motility and dissemination to neighbouring cells. The project CAGECONTROL aims to further understand the process of formation of septin cages. Hosted in Prof. Serge Mostowy lab at the London School of Hygiene and Tropical Medicine, the projet “CAGECONTROL: Identification and manipulation of factors that control Shigella flexneri entrapment in septin cages” had two main objectives: a) What is the breadth of factors that determine septin-cage formation? and b) Can we manipulate septin cages to control the fate of S. flexneri during infection?
Data: CORDIS, © European Union
Project 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.
Original text from CORDIS.
Participants
- LONDON SCHOOL OF HYGIENE AND TROPICAL MEDICINE ROYAL CHARTER · LondonCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union