Intracellular niche formation of Shigella

Supervisor Organisation PhD Awarding Entity: phd location
Institut Pasteur
Université de Paris Cité
Institut Pasteur, Paris, France

Research Focus

This project investigates how Shigella, the causative agent of human dysentery, invades enterocytes of the human colon. In particular, we will address how the pathogen uses a molecular weapon called the type 3 secretion system to subvert host pathways and compartments to model its intracellular niche. During these steps, in situ formed infection associated macropinosomes interact with the pathogen compartment that is called the bacterial containing vacuole. We will decipher this dynamic crosstalk using the advanced imaging approaches of the CLEXM consortium.

Cryo-SXT will provide information about the cellular context around the infection site, and the data will be correlated with cryo-ET data. It will bridge the information and resolution gap between cryo-fluorescence microscopy and cryo-ET, providing a detailed 3D view of the remodelling of cellular architecture upon infection. Nanoscale detail of the whole cellular context around the infectious event, combined with the high-resolution structural information from cryo-ET on the same sample/grid will be obtained. We will further implement analytical workflows to extract quantitative information on the compartments subverted by the pathogen during the invasion process.

Methodology

This project uses cellular models to investigate the infection of enterocytes by Shigella. Fluorescence imaging provides information on the molecular factors involved in the host pathogen crosstalk. Both, confocal and super-resolution techniques are used. This will be correlated with electron microscopy in cryo conditions, large volume EM, as well as SXT.

Aim 1

We will use fluorescence biosensors to pinpoint the successive invasion steps of Shigella. Sensors will identify pathogen factors, as well as subverted host proteins and their modulation by the pathogen.

Aim 2

The fluorescence biosensors will be exploited within correlative workflows at large volumes (either by FIBSEM or SXT) and at high rsolution by cryo tomography.

Aim 3

Bioinformatics workflows will be used and developed to decipher the molecular crosstalk for the intracellular niche formation of Shigella

Pictures Attached

 Image description: A workflow for correlative light and electron microscopy to investigate the intracellular niches of Shigella. Samples are prepared for optical imaging on coverslips with an engraved coordinate system. Coordinates can be identified in the different imaging modalities, and are used to correlate the images from fluroecence and electron microscopy.

 

PhD Researcher

Name: Keith Egger

University: Université Paris-Cité

Supervisor’s Name: Dr. Anastasia Gazi/ Dr. Jost Enninga 

Keith Egger completed his Bachelor’s in Biochemistry with a focus on X-ray crystallography at the University of Victoria in 2020. Following this, he spent two years as a research assistant at Harvard Medical School in the laboratory of Dr. Marcia Goldberg, where he delved into understanding the role of Shigella effectors in infection, as well as exploring the innate immune response to bacterial pathogens. He successfully defended his master’s thesis in immunology in 2023 at Dr. Charlotte Odendall’s lab at King’s College London, with his project concentrating on the modulation of interferon by Salmonella effectors. Currently, he has embarked on his Ph.D. journey in Dr. Jost Enninga’s lab at the Institut Pasteur, focusing on the study of Shigella-host interactions using Cryo-CLEM pipelines.

His research interests lie in the cell biology of infection. Through the application of multiscaler imaging techniques to the field of infection biology and integration of powerful techniques in fluorescence microscopy, cryo-ET, and SXT, he can gain insight into how bacterial pathogens impact host physiology. His project focuses on deciphering how enteric bacteria utilize their secretion systems to facilitate infection, studying the dynamics of entry and vacuolar escape from a whole cell to a single-molecule level.

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